Resveratrolün sıçan random deri fleplerinin yaşayabilirliği üzerine etkisi: Deneysel çalışma
The effect of resveratrol on the survival of random skin flaps in rats: An experimental study
Yazar: ALİ CEM AKPINAR
Danışman: DOÇ. DR. SELMA SÖNMEZ ERGÜN
Bezm-i Alem Vakıf Üniversitesi / Tıp Fakültesi / Plastik Rekonstrüktif ve Estetik Cerrahi Ana Bilim Dalı
Derece: Tıpta Uzmanlık, 2011
Konu: Plastik ve Rekonstrüktif Cerrahi = Plastic and Reconstructive Surgery
Dizin: Cerrahi flepler = Surgical flaps ; Deri = Skin ; Deri nakli = Skin transplantation ; Resveratrol = Resveratrol ; Serbest radikaller = Free radicals ; Sıçanlar = Rats ; Yaşama gücü = Vitality

Özet:
Random paternli cilt flepleri doku defektlerinin kapatılması amacıyla plastik cerrahide sık kullanılır. Flep boyu uzadıkça, flep vaskülaritesini etkileyecek bir travma varlığında ve/veya flep planlamasının hatalı yapıldığı durumlarda flebin kan akımı kritik düzeylere düşebilir. Flep yaşayabilirliğini arttırmak için sempatolitik, direkt vazodilatatör, antitrombotik ve antikoagülan ajan kullanımı, kanın reolojik özelliklerini değiştirme, antioksidan ilaçlar kullanılması gibi yaklaşımlar mevcuttur. İskemiye maruz kalan bölgede serbest oksijen radikalleri ve nötrofil birikimi doku hasarına neden olur. Oluşan toksisiteyi önlemek için bir çok çalışma yapılmıştır. Resveratrol, bitkiler tarafından travmatik zedelenme veya fungal saldırılara karşı sentezlenir. Yapılan çalışmalarda resveratrolün anti-agregan, anti-oksidan, anti-enflammatuar ve vazodilatasyon etkileri olduğu bildirilmiştir. Bu etkileri göz önüne alınarak resveratrolün flep yaşayabilirliğini arttıracağı düşünülerek çalışma planlandı. Resveratrolün flep yaşayabilirliğini olumlu yönde etkilediğinin gösterilmesi ile ilacın insana uygulanması mümkün olacaktır.Bu çalışma İstanbul Üniversitesi Deneysel Tıp Araştırma Enstitüsü'nde yapıldı. Deney hayvanı olarak 24 adet Sprague-Dawley cinsi erişkin dişi sıçan kullanıldı. Sıçanlar 8'er adetten oluşan üç gruba ayrıldı. Sıçanlara flep kaldırma işlemi uygulandı. İlk dozu cerrahi işlemden 1 saat önce olmak üzere toplam 7 gün boyunca intraperitoneal yoldan günde 1 kez verilmek üzere 1.gruba izotonik NaCl, 2.gruba Dimetilsülfoksit(DMSO), 3.gruba DMSO'da çözdürülmüş 1 mg/kg resveratrol enjekte edildi. Postoperatif 7. günde sıçanların sırt bölgesindeki flepler dijital fotoğraf makinesi ile fotoğraflandı. Nekroz alanının flep boyutuna oranı Adobe Photoshop CS2 programı ile hesaplandı. Postoperatif 7. günde doku örnekleri alındı ve bütün hayvanlara ötenazi uygulandı. Histopatolojik incelemede birim alandaki kapiller ve enflamatuar hücre sayımı yapıldı. Sonuçlar Mann Whitney U testi ve ANOVA Varyans analizi ile değerlendirildi.Bütün sıçanlar deney sonuna kadar canlı kaldı. Operasyon sonrası 7. günde bütün fleplerde nekroz hattı belirgin şekilde oluştu. Flep canlı alanı pembe-beyaz renkte ve normal tonusta iken nekroz bölgesi siyah renkte ve sert idi. Postoperatif 7. gün yapılan yaşayabilirlik değerlendirmesinde resveratrol grubunun yaşayan flep oranının kontrol gruplarına göre istatistiksel olarak anlamlı şekilde yüksek olduğu görüldü. Histopatolojik olarak da resveratrol grubunda kontrol gruplarına göre istatistiksel olarak anlamlı derecede birim alanda daha fazla kapiller, daha az enflammatuar hücre olduğu bulundu.Çalışmamızda preoperatif başlanan ve postoperatif 7 gün boyunca devam edilen 1 mg/kg sistemik resveratrol uygulamasının flep yaşayabilirliğini arttırdığı gösterilmiştir. Flep cerrahisi planlanan hastalara operasyon öncesi ve sonrası sistemik resveratrol uygulaması ile komplikasyonların, hastanede yatış süresinin, ek cerrahi girişimlerin azalacağı düşünülmektedir.

Abstract:
Skin flaps are usually made for the primary coverage of a large bare area of the body resulting from skin and tissue defects. In skin flap surgery, distal ischemic necrosis is the most important complication encountered. Various pharmacologic agents have been investigated for their efficacy in preventing or reversing skin flap ischemia. Anticoagulants, vasodilators, glucocorticoids, free radical scavengers, sympatholytics, prostaglandin inhibitors, calcium channel blockers, and hemorheologic agents are among the drugs thought to be beneficial for survival. One alternative approach, surgical delay, has been widely used to improve the survival of ischemic skin flaps. However, this approach has the disadvantage of being a two-stage procedure, which causes increased surgical invasiveness and complications.Resveratrol (trans-3,4?,5-trihydroxystilbene) is a polyphenol found in various fruits and vegetables, and is abundant in grapes. The possible mechanisms for its pharmacological activities involve modulating lipid metabolism, platelet aggregation, and the inflammatory response, including downregulation of pro-inflammatory mediators, alteration of eicosanoid synthesis, or inhibition of activated immune cells, primarily represented by neutrophils and monocytes/ macrophages. Furthermore, resveratrol is a more powerful antioxidant than vitamin E in preventing low-density lipoprotein oxidation, and is considered as a possible therapeutic agent in treating acute ischemia ? reperfusion (IR) injury.The purpose of this study was to determine whether resveratrol would improve the survival of random-pattern skin flaps in a rat model.Materials MethodsThis study was conducted in Experimental Medical Research Institute of Istanbul University after the approval obtained from the Animal Care Committee of the Istanbul University, and the animals were treated according to the experimental protocols under its regulations.Twenty-four Sprague-Dawley rats, weighing between 220 and 250 g, were housed separately, at room temperature (23±1°C), humidity of 55±5 percent, a light/dark cycle of 12 hours, and provided chow and water ad libitum. All procedures were conducted in accordance with the Declaration of Helsinki and the Guide for the Care and Use of Laboratory Animals.The animals were divided into three groups of 8 animals each. The first group (group 1) was the control group (intraperitoneal isotonic NaCl injection), and the second group (group 2) was intraperitoneally injected with Dimethylsulphoxide (DMSO), and the third group (group 3) was intraperitoneally injected with resveratrol in DMSO.Assessment of Improvement of Flap SurvivalSacrification was performed by cervical dislocation 7 days after flap elevation. After gross observation of the skin flaps, zones of dark color and those covered with scabs were defined as the necrotic area, and the other areas of the skin flaps were defined as the surviving area. First, the areas of the flaps were photographed with a digital camera (Sony DSC-N2) and were recorded on a computer. 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) by using a computer software program (Adobe Photoshop CS2). Finally, The survival rate of the flap was expressed as the percentage of the surviving area compared with the total area of the flap. Survival rate =(surviving area/total area) x100.ResultsInfection and mortality were not observed during our experimental study. We observed the gross appearance in the control group daily after operation. On day 1, there were no obvious changes. Various degrees of epidermolysis appeared 2 to 3 days after surgery. At day 4, necrosis began to appear clearly in the distal portion of the skin flap, and 7 days after surgery, a necrotic area was clearly defined with apparent demarcation in all skin flaps.On day 7, a clear demarcation between the surviving and necrotic regions was observed in the skin flaps in the control and the experimental groups. The surviving region appeared soft, with a normal pink color. In contrast, the necrotic region appeared rigid and had a dark color. The mean percentage of survival of flaps was 48±4.8%, 48±4.1%, and 82±4.9%, respectively. There was no statistically significant difference between the mean survival rates of the control group and group 2. The mean survival rate of group 3 was significantly higher than that of control group and group 2 (p<0.01).On Hematoxylin-Eosin staining, necrosis was pathologically confirmed in the control group and the group 2, a marked increase of neutrophils adhered to the endothelium was observed. On CD31 staining, microvessels were barely detected, even in the seemingly viable area of these groups. In contrast, in the group 3, cells maintained their normal morphologies, with very little evidence of necrosis, adherent neutrophils were found to a lesser extent, and a greater number of microvessels were observed.Inflammatory cell number per 1mm2 for individual rats of the groups are presented in Table 2. The mean number of the inflammatory cells per 1 mm2 was 25±3.8, 23±3.0, and 8±0.7, respectively. There was no statistically significant difference between the mean number of inflammatory cells of the control group and group 2. It showed a significantly higher average number of inflammatory cells in group 3 compared with the control group and group 2 (p<0.01).Microvessel number per 1 mm2 for individual rats of the groups are presented in Table 3. The mean number of the microvessels per 1 mm2 was 20±2.6, 21±2.8, and 40±3.4, respectively. There was no statistically significant difference between the mean number of microvessels of the control group and group 2. The mean number of microvessels in group 3 was significantly higher than that of control group and group 2 (p<0.01).DiscussionSkin flap necrosis remains a significant problem in plastic and reconstructive surgery. Despite the new flap designs and surgical technique, ischemic necrosis is still a complication. The morbidity from flap loss can be substantial and can result in increased costs resulting from prolonged hospital stay, the need for additional surgery, and an increased number of outpatient visits.According to Rohrich et al., an ideal pharmacological agent for improving flap survival would have the following features: clinical availability, easy administration, a high therapeutic index (safety), reproducibility of effective results, feasibility of only postoperative treatment, cost-effectiveness, known mechanism of action, established bioavailability, and protective effects on flap necrosis. To date, no substance has fulfilled all of the requirements and an ideal substance has not yet been found. Several antioxidants protecting the tissues against the damage of free radicals such as melatonin, ?- tocopherol, ascorbic acid and carnitine and Nacetylcysteine have proven to be efficient in attenuating the changes of ischemia-reperfusion injury. Resveratrol (3.4?, 5 trihydroxytransstilbene),has been shown to regulate many biological activities. Resveratrol has been reported to be protective against atherosclerosis by exerting antioxidant activity and to protect rat heart from I/R injury. Moreover, resveratrol was considered to have anticancer and anti-inflammatory actions. Resveratrol was found to be an effective scavenger of hydroxyl and superoxide, and to exhibit a protective effect against lipid peroxidation in cell membranes and DNA damage caused by free radicals.To study distal ischemia, an appropriate ischemic skin flap model is necessary. A caudally based 10x3 cm dorsal rat flap was advocated by Khouri and coworkers, McFarlane et al.. They reported this model as the most suitable choice for experimental flap studies. Several authors have used this flap model for their investigations. As for the advantages of this ischemic skin flap model, they are that it is easy to make, and the obvious distal necrosis in the control group (approximately 50 percent) can be easily observed. We also used the same flap model in our study. The areas of flap necrosis in our control group were found to be similar to those of control groups of other studies.The improvement of the flap survival by resveratrol may be due to its anti-oxidant and anti-inflammatory effects against ischemia reperfusion injury. Vasoconstriction, edema, leukocyte activation/aggregation that induce flap necrosis after ischemia could be prevented by resveratrol. Resveratrol also increases microcirculation through inhibition of perivascular and intravascular leukocyte aggregation. Low number of inflammatory cells in the biopsy specimens of the resveratrol group in this study could be explained by the anti-inflammatory effect of resveratrol. Anti-inflammatory effect results from the nitric oxide synthase and cyclooxigenase pathways. Resveratrol is angiostatic, but microvessels were found to be increased in number histopathologically in resveratrol group. This paradox may be explained by the increase of nitric oxide and vascular endothelial growth factor levels by resveratrol.This study showed that administration of resveratrol preoperatively and 7 days postoperatively could improve the survival of ischemic skin flaps. On the basis of our findings in this study, we think that resveratrol might have important therapeutic potential in improving skin flap survival in clinical settings.Our present study is one of the first attempts to examine the therapeutic potential of resveratrol in improving skin flap survival in a model. Future efforts should be directed at defining optimal drug doses and application intervals.