Protective effect of melatonin on random pattern skin flap necrosis in pinealectomized rat

Authors


Address reprint requests to Ali Gurlek, Department of Plastic and Reconstructive Surgery, Medical Faculty, Inonu University, T Ozal Medical Center, TR 44069 Malatya, Turkey.
E-mail: agurlek@hotmail.com

Abstract

Abstract: Random pattern skin flaps are still widely used in plastic surgery. However, necrosis in the distal portion resulting from ischemia is a serious problem, increasing the cost of treatment and hospitalization. Free oxygen radicals and increased neutrophil accumulation play an important role in tissue injury and may lead to partial or complete flap necrosis. To enhance skin flap viability, a variety of pharmacological agents have been intensively investigated. The aim of this study is to test the effects of melatonin, the chief secretory product of the pineal gland and a highly effective antioxidant, on random pattern skin flap survival in rats. Herein, to investigate the physiological and pharmacological role of melatonin on dorsal skin flap survival. Pharmacological (0.4, 4 and 40 mg/kg) levels of melatonin were given intraperitoneally (i.p.). For this, pinealectomized (Px) and sham operated (non-Px) rats were used. The effects of melatonin on levels of malondialdehyde (MDA), nitric oxide (NO), glutathione (GSH) and the activities of glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) were measured in the skin flap. The ratio of skin flap necrosis was compared among the experimental groups by using planimetry. MDA and NO levels were found to be higher in Px than non-Px rats; while GSH levels and GSH-Px, and SOD activities were reduced. Melatonin administration to Px rats reduced MDA and NO levels and increased GSH, GSH-Px, SOD levels. Melatonin also reduced the ratio of flap necrosis determined by using planimetry and supported through the photography. In conclusion, these results show that both physiological and pharmacological concentrations of melatonin improve skin flap viability.

Introduction

Random pattern skin flaps are still widely used as a reconstructive option in plastic surgery. An adequate blood supply is crucial for survival of these flaps, and any injury to flap vascularity or a too-risky flap design causes ischemia and may lead to partial or complete flap necrosis. Surgical delay, which is known to enhance flap viability, is an effective technique that is often used for this purpose [1]. However, this approach has the disadvantage of involving a two-stage surgical procedure. Various pharmacological agents including sympatholytics, vasodilators, calcium channel blockers, hemorheological agents, prostaglandin inhibitors, anticoagulants, glucocorticoids and free radical scavengers are among the drugs thought to be beneficial for flap survival and they have been investigated for their efficacy in preventing or reversing skin flap ischemia [2].

The clinical use of random pattern flaps may be limited by the development of necrosis in the distal area because of ischemia–reperfusion (I/R) injury. The effects of I/R on random pattern skin flap have been documented in numerous species of experimental animals. One leading mechanism of this toxicity is believed to involve reactive oxygen radical species (ROS) generation and neutrophil accumulation. Based on this relationship, treatment of the flaps with either exogenous superoxide dismutase (SOD) [3], catalase [4], allopurinol [5] vitamin C [6], dexamethasone [7], heparin [8], or deferoxamine [9] significantly increase survival.

Recently, Parlakpinar et al. [10] reported that physiological and pharmacological concentrations of melatonin in pinealectomized (Px) and non-pinealectomized rats were important in preventing the nephrotoxicity resulting from antineoplastic agent cisplatin. Considering the reduced oxidative damage because of melatonin treatment, the authors attributed melatonin's protective actions to its direct and indirect antioxidant activity. The pineal secretory product, melatonin, is known to exhibit free radical scavenging ability and reduces neutrophil accumulation [11]. Melatonin also activates several antioxidative enzymes including glutathione peroxidase (GSH-Px), modulates gene expression for several protective enzymes and reduces lipid peroxidation [12, 13]. While the beneficial effects of melatonin in different tissues have been investigated, to date no one has investigated the protective effect of physiological and pharmacological concentrations of melatonin against necrosis in the random pattern skin flap in Px rats.

This study was designed to investigate the effects of physiological and pharmacological concentrations of melatonin on the random pattern skin flap in a Px rat model. For this study, Px and sham operated (non-Px) rats were used. The effects of pharmacological doses (0.4, 4 and 40 mg/kg) exogenous melatonin administration were also determined. As it has been reported that pinealectomy may cause hypertension for the first 60 days [14], we used rats that were Px 2 months before flap surgery to eliminate any possible effect of pinealectomy-induced hypertension.

Materials and methods

Experimental conditions

Female Wistar rats weighing 150–200 g were housed in temperature (21 ± 2°C) and humidity (60 ± 5%) controlled room in which a 12:12 hr light:dark cycle was maintained.

Rats with Px or non-Px were housed for 2 months before the beginning of all injections.

Pinealectomy

Pinealectomy was performed as described by Hoffman and Reiter [15]. Rats were preoperatively anesthetized by intraperitoneal (i.p.) application of a mixture consisting of ketamin hydrochloride (75 mg/kg) and xylazine hydrochloride (8 mg/kg). The entire procedure was completed within 15 min. Px was confirmed by histological evaluation of the gland for each animal.

Forty-four female Wistar rats were divided into seven groups: Group 1 (sham; n = 6) was treated only vehicle (i.p.). Group 2 (flap elevated control group; n = 7) was treated (i.p.) with vehicle only. Group 3 (Px group; n = 6) was treated only vehicle. Group 4 (flap elevated Px group; n = 7) was treated with vehicle only. Group 5 (flap elevated Px group; n = 6) was treated with 0.4 mg/kg Mel 1 hr before skin surgery and continued for 6 days after the flap elevation. Group 6 (flap elevated Px group; n = 6) was treated with 4 mg/kg Mel 1 hr before skin surgery and continued for 6 days. Group 7 (flap elevated Px group; n = 6) was treated with 40 mg/kg Mel 1 hr before skin surgery and continued for 6 days. Intraperitoneal route for diluent and melatonin administration was chosen because of the ease of application and the reproducibility of dosing.

The skin of the dorsal trunk was shaved with electric clippers and then prepared with Betadine® (Poviiodeks, Kim-Pa Corporation, Istanbul, Turkey). During the surgical procedure, aseptic conditions were maintained by providing a local sterile environment. A dorsal random pattern skin flap with a size of 10 × 3 cm was elevated on the dorsal trunk of the rats according to the method described by Khouri et al. [16] with meticulous hemostasis; the flap was sutured back into its place with 4-0 running nylon sutures (Fig. 2A). Animals in the experimental group were treated with either 0.4, 4 or 40 mg/kg pharmacological concentrations of melatonin per day for 6 days.

Figure 2.

Size of the standard rat dorsal skin flap used in the current study (A); flap elevated sham-operated (non-Px) group (B); note the tissue necrosis in black; flap elevated pinealectomized (Px) group (C) the necrosis ratio is higher than in the non-Px animals; flap elevated Px group treated with 0.4 mg/kg melatonin (D); flap elevated Px group treated with 4 mg/kg melatonin (E); flap elevated Px group treated with 40 mg/kg melatonin (F).

Flap viability was evaluated 7 days after the initial operation, at which time a certain amount of necrosis in the distal part of all dorsal flaps was noted (Fig. 2A–F). On day 7, the rats were reanesthetized for evaluation of flap viability. All groups were photographed and then, the necrotic skin (defined by the necrotic skin borders) and total flap (defined by the surgical borders) areas were delineated, and surface areas were calculated (in square centimeters) using computer-assisted planimetry. The necrotic surface area was divided by the total flap area, and the results are expressed as percentages of skin necrosis (Fig 1). The animals were then killed. The skin biopsy was taken from an area between 3 and 4 cm proximal of flap to determine the levels of malondialdehyde (MDA), nitric oxide (NO) and glutathione (GSH) levels and GSH-Px and SOD activities.

Figure 1.

The effects of pinealectomy or melatonin (Mel) administration on infarct size/flap size ratio in pinealectomized (Px) or sham-operated (non-Px) rats with flap elevation. aP < 0.05 versus non-Px + flap group, bP < 0.05 versus Px + flap group.

Melatonin® (Sigma Chemical Co., St Louis, MO, USA), used in this study, was dissolved in ethanol and diluted in saline to give a final concentration of 5% ethanol.

All experiments in this study were performed in accordance with the guidelines for animal research from the National Institutes of Health and were approved by the Committee on Animal Research at Inonu University, Malatya.

Biochemical determinations

One hundred milligrams of frozen flap tissue biopsy specimens, cut into pieces on dry ice, were homogenized in 1.15% KCl buffer (1:9, w/v) using a manual glass homogenizer for approximately 5 min and flushed by centrifugation for approximately 10 s to remove large debris. The supernatant was used for analysis.

The MDA content of homogenates was determined spectrophotometrically by measuring the presence of thiobarbituric acid reactive substances [17]. Results are expressed as nmol/g tissue.

As tissue nitrite (NOinline image) and nitrate (NOinline image) levels can be used to estimate NO production, we measured the concentration of these stable NO oxidative metabolites. Quantitation of NOinline image and NO3 was based on the Griess reaction, in which a chromophore with a strong absorbance at 545 nm is formed by reaction of NOinline image with a mixture of naphthlethylenediamine and sulfanilamide [18]. Results are expressed as μmol/g tissue.

Glutathione was determined by the spectrophotometric method which was based on the use of Elman's reagent [19]. Results are expressed as nmol/mg tissue.

The GSH-Px activity was measured by the method of Paglia and Valentina [20]. In the presence of glutathione reductase (GSH-Rd) and nicotinamide adenine dinucleotide phosphate (NADPH) the oxidized GSH is immediately converted to the reduced form with a concomitant oxidation of NADPH to NADP+. The decrease in absorbance at 340 nm is measured. GSH-Px activity was expressed as U/g protein.

The SOD enzyme activity determination was based on the production of H2O2 from xanthine by xanthine oxidase and reduction of nitroblue tetrazolium as previously described [21]. The product was evaluated spectrophotometrically. Results are expressed as U/g protein.

Statistical analysis

Flap tissue MDA, NO, GSH, GSH-Px, and SOD levels were analyzed by one-way ANOVA. Post hoc comparisons were performed using Tukey's test. Differences were considered significant when P < 0.05. Results are expressed as mean ± S.E.M.

Results

Table 1 summarizes the data obtained on the effects of Px and treatment of melatonin (0.4, 4, 40 mg/kg) on tissue MDA, NO, GSH, GSH-Px and SOD levels. MDA, NO levels were found to be higher in Px group than non-Px group while GSH levels and GSH-Px and SOD activities were reduced as a result of tissue injury. Furthermore Px + flap elevation also caused an increased MDA, NO levels and significantly reduced GSH, GSH-Px and SOD values compared with those in Px rats alone.

Table 1.  The effects of pinealectomy or melatonin (Mel) administration on tissue components and enzyme activities in pinealectomized (Px) or sham-operated (non-Px) rats with or without flap elevation
GroupsMDA (nmol/g tissue)NO (μmol/g tissue)GSH (nmol/g tissue)GSH-Px (U/g protein)SOD (U/g protein)
  1. aP < 0.05 versus sham group.

  2. bP < 0.05 versus Px group.

  3. cP < 0.05 versus Px + flap group.

Sham29.4 ± 1.940.9 ± 3.87.8 ± 0.3381 ± 7110.8 ± 6.5
Non-Px + flap70.7 ± 0.8a116.5 ± 2.7a1.6 ± 0.2a168 ± 5a27.9 ± 0.6a
Px77.3 ± 1.3a126.3 ± 1.5a2.3 ± 0.2a197 ± 4a60.3 ± 2.6a
Px + flap99.3 ± 3.3 b153.0 ± 4.9b0.9 ± 0.1b122 ± 3b18.7 ± 2.4b
Px + flap + Mel (0.4)72.3 ± 1.981.4 ± 3.3c1.9 ± 0.1c161 ± 2c36.6 ± 1.2c
Px + flap + Mel (4)56.4 ± 1.1c61.4 ± 2.8c2.9 ± 0.1c224 ± 4c46.4 ± 4.4c
Px + flap + Mel (40)49.7 ± 2.4c63.8 ± 3.7c4.9 ± 0.2c267 ± 7c74.5 ± 4.1c

Melatonin, given to Px rats at doses of 4 and 40 mg/kg significantly reduced MDA levels whereas at dose of 0.4 mg/kg no significant change was measured. However, all doses of melatonin when given to Px rats caused significantly reduced NO levels and significantly increases in GSH, GSH-Px and SOD levels.

Table 2 summarizes the infarct size/flap size ratio. Px + flap elevation caused a significant increase in infarct size expressed as the percentage of risk zone (48.9 ± 2.4) when compared with non-Px + flap elevated animals (37.3 ± 1.7).

Table 2.  The effects of pinealectomy or melatonin (Mel) administration on infarct size/flap size ratio in pinealectomized (Px) or sham-operated (non-Px) rats with flap elevation
GroupsInfarct size/flap size (%)
  1. aP < 0.05 versus non-Px + flap group.

  2. bP < 0.05 versus Px + flap group.

Non-Px + flap37.3 ± 1.7
Px + flap48.9 ± 2.4a
Px + flap + Mel (0.4)37.5 ± 5.3
Px + flap + Mel (4)29.8 ± 1.7b
Px + flap + Mel (40)29.2 ± 1.8b

Melatonin administration (4 and 40 mg/kg) to Px rats significantly reduced the infarct size ratio (29.8 ± 1.7 and 29.2 ± 1.8 respectively). Although 0.4 mg/kg melatonin did not reach statistical significance, it tended to reduce the infarct size/flap size (37.5 ± 5.3). This data indicated that the ratio of infarct size/flap size in 0.4 mg/kg melatonin treated rats was similar to that in non-Px + flap group (37.3 ± 1.7 and 37.5 ± 5.3). This results show that there is difference between the 0.4 mg/kg melatonin treated rats and the Px + flap group (37.3 ± 1.7 and 48.9 ± 2.4). Also Fig. 2 clearly shows that increase in melatonin concentrations had increasing inhibitory effects on skin flap necrosis. Thus panel D (0.4 mg/kg) has more necrosis than panel E (4 mg/kg) and panel E has more necrosis than panel F (40 mg/kg). This appear to be a beautiful dose–response relationship.

Discussion

Although random pattern skin flaps are still widely used in plastic surgery, necrosis in the distal portion of the flaps resulting from ischemia is a serious problem leading to an increase in treatment expenses and hospitalization. An adequate blood supply is crucial for survival of these flaps, and any injury to flap vascularity, a too-risky flap design or I/R injury may lead to partial or complete flap necrosis. To overcome this potential problem, investigators have focused on the improvement of flap survival, especially among high-risk patients. In these studies, it has been demonstrated that vasoconstriction, edema formation, accumulation and activation of leukocytes contributed to formation of skin flap necrosis [22]. It is obvious that these factors must be controlled and prevented to achieve optimal flap survival.

There are many studies concerning the role of ROS and neutrophil accumulation in the pathophysiology of skin flap necrosis. ROS have been implicated in a wide range of biological functions, but they can express both beneficial and highly toxic effects on cellular homeostasis [23]. Several conditions are known to disturb the balance between the production of ROS and cellular defenses resulting in dysfunction and cellular destruction [24].

The ROS play an important role in I/R injury pathogenesis. When tissue oxygen levels decrease, the intracellular metabolism changes from aerobic to anaerobic. In this situation, lactate accumulates, the pH of the cell drops, and availability of ATP which reduces fuel in ionic pumps. These changes impair membrane transport functions. The formation of oxygen free radicals begins when oxygen is reintroduced to tissues after ischemia. Thus, the fresh supply of oxygen, which accompanies reperfusion, generates oxygen free radicals [25] which are toxic to all biologic molecules, including proteins, polysaccharides, nucleic acids, collagen, fatty acids, phospholipids, etc. Within the microcirculation, endothelial cell damage results in exposure of collagen and basement membranes, promoting adhesion of platelets and granulocytes, and initiating the cascade of microcirculatory thrombosis [26].

Free radical scavengers are compounds which neutralize oxygen-bared reactants and into generate relatively harmless by-products. Scavengers include SOD [3], catalase [4], GSH [27], deferoxamine [9], allopurinol [5], dexamethasone [7], heparin [8], caffeic acid phenethyl ester [28], aminoguanidine [29], vitamin C [6], vitamin E [30], melatonin [31], etc. All these free radical scavengers had been shown to be effective experimentally, but whether they will be useful clinically has yet to be determined. Clinical trials in the settings of sepsis syndrome, myocardial infarction, organ transplantation and cardiopulmonary bypass have been accomplished and some show promising effects for oxygen radical scavengers [32, 33].

The importance of the neutrophil in I/R injury has been recognized for many years. Increased production of oxygen free radicals and infiltration of neutrophils into tissue subjected to I/R have emphasized that neutrophils play a direct role in the development of injury. A significant infiltration of neutrophils into skeletal muscle [34], skin flaps [35], bowel [36] and liver [37] subjected to I/R injury has also been demonstrated in animal models. Besides free radicals, neutrophils also produce proteinases and phospholipases, which damage endothelial cells and vascular membranes leading to tissue edema and further thrombosis [38]. To prevent neutrophils accumulation and to improve the survival length of flaps many pharmacological and therapeutic manipulations have been studied intensively [39–41].

Till date, no ideal substance has been found to reduce skin flap necrosis. As melatonin is easily administered and absorbed, its administration leads to effective therapeutic tissue levels with significant systemic drug distribution, with a high therapeutic index (safety). Furthermore, it could be easily administered postoperatively even as an outpatient treatment in clinical settings.

The pineal secretory product, melatonin, is a documented scavenger of free radicals including the hydroxyl radical, singlet oxygen, peroxyl radical, and the peroxynitrite anion and, besides scavenging free radicals, melatonin also may reduce their generation [42]. Additionally, melatonin's antioxidant actions probably derive from its stimulatory effect on SOD, GSH-Px, GSH-Rd, and glucose-6-phosphate dehydrogenase and its inhibitory action on NO synthase [12, 13]. In the current study, Px caused NO elevation and all doses of melatonin to Px rats significantly reduced NO levels. Melatonin is highly lipophilic and it passes easily through biological membranes [43]. This is an advantage for melatonin over some other antioxidants which penetrate cells more slowly. Likely the property of melatonin to rapidly enter cells may be an important feature in reducing flap necrosis. On the contrary, the role of physiological levels of melatonin, which are known to decrease with age [44], in the prevention of this oxidative damage has been tested in a number of studies [45]. The night-time blood melatonin peak is eliminated after Px [46].

Previous studies pointed to the beneficial antioxidant and free radical scavenger effects of melatonin in protecting against neutrophil accumulation. Many investigators including Agapito et al. [47] and Parlakpinar et al. [10] reported that melatonin attenuates lipid peroxidation and studies have also shown the increase to GSH levels [48].

Malondialdehyde, product of lipid peroxidation, is generated as a result of toxic effects of active oxygen radicals which destroy unsaturated fatty acids in the cell membranes [10]. Although tissue MDA levels are clearly decreased by melatonin, its mechanism is not clear. Melatonin may directly eliminate free oxygen radicals or directly increase the antioxidant enzyme activity and prevent the inhibition of these enzymes. Also melatonin has been shown to react with NO· [49] generated during the decomposition of 1-hydroxy-3-oxo-3 (N-methyl-3-aminopropyl)-3-methyl-1-triazene in a cell-free system. These findings are in accordance with those of Mahal et al. [50] who also found that melatonin can detoxify NO., with a bimolecular rate constant of 3.0 × 107/m/s. Recently, Blanchard et al. [51] showed that melatonin was able to interact with NO·, but only in the presence of O2, a finding suggesting that melatonin reacts with NO-derived nitrogen species (NOx), possibly ONOO. The effect of NO on the microcirculation in the periphery of a flap remains unclear, and its effect on flap survival is also unknown because NO has a dual action.

The enzyme GSH-Px utilizes GSH, an intracellular thiol that is typically in millimolar concentrations, as a substrate. Maintaining high intracellular concentrations of GSH also seems to be a function of melatonin as this indole stimulates the activity of its rate-limiting enzyme, gamma-glutamylcysteine synthase [52]. When GSH is metabolized by GSH-Px, a reaction that also requires H2O2 or other hydroperoxides, it is converted to oxidized glutathione (GSSG). Within cells the GSH:GSSG ratio is normally greatly in favor of the former, and to maintain this ratio GSSG is rapidly metabolized back to GSH by GSH-Rd. As noted above, experimental evidence has shown that melatonin also promotes the activity of GSH-Rd thereby helping to maintain high levels of reduced GSH [53].

In the current study, Px animals had the lowest GSH and GSH-Px levels. All doses of melatonin were found to have, beneficial effects on GSH levels and metabolism.

Considering the reduced oxidative damage because of melatonin treatment, melatonin's protective actions in the current study are believed to be a consequence of direct and indirect antioxidative activities. Our results are consistent with the literature that melatonin administration reduces MDA and NO levels in Px rats and increases GSH levels and GSH-Px, SOD activities. Also, pharmacological concentrations of melatonin reduce the ratio of skin flap necrosis which, herein was determined by using planimetry and supported by photography. These findings strongly suggest that both physiological and pharmacological concentrations of melatonin are important in reducing injury in random pattern dorsal skin flap tissue.

In conclusion, the current findings indicate that melatonin supplementation may reduce skin flap injury especially in individuals with low plasma melatonin concentrations. We feel that melatonin could be effectively combined with flap surgery, especially in older patients. Finally, the protective effects of both physiologic and pharmacological concentrations of melatonin on the flap survival should also be further investigated.

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