RESEARCH
Chlorogenic Acid Enhances Abdominal Skin Flap Survival Based on Epigastric Artery in Nondiabetic and Diabetic Rats Deniz Bagdas, DVM, PhD,* Betul Cam Etoz, MD, PhD,Þ Zulfiye Gul, MS,þ Musa Ozgur Ozyigit, DVM, PhD,§ Nilufer Cinkilic, PhD,|| Sevda Inan, DVM, MS,§ Naciye Isbil Buyukcoskun, PhD,Þ Kasim Ozluk, PhD,Þ and Mine Sibel Gurun, MD, PhDþ Abstract: Previous studies showed that chlorogenic acid (CGA) accelerates wound healing via its antioxidant activity. We aimed to investigate the effect of CGA in an experimental epigastric abdominal skin f lap model in nondiabetic and diabetic rats. Rats were firstly divided into 2 groups: nondiabetic and diabetic. Diabetes was induced by streptozotocin. Then, 4 subgroups were created for each group: vehicle as well as 0.2 mg/0.5 mL, 1 mg/0.5 mL, and 5 mg/0.5 mL CGA treatments. Right epigastric arteryYbased abdominal skin f laps were elevated and sutured back into their original position. Chlorogenic acid or vehicle was injected once into the femoral arteries by leaving the epigastric artery as the single artery feeding the f laps during the injection. On postoperative day 7, f lap survivals were evaluated, and the rats were killed. Distal f lap tissues were collected for histopathological and biochemical assays. Chlorogenic acid showed greater f lap survival in both nondiabetic and diabetic rats. Capillary density was increased, and necrosis was reduced in the CGA-treated rats. Chlorogenic acid decreased malondialdehyde levels as well as increased reduced glutathione and superoxide dismutase levels in the f lap tissues. This study showed that CGA significantly improved f lap survival by its antioxidant activities with intra-arterial local injections. Key Words: antioxidant, chlorogenic acid, diabetes, epigastric artery, flap survival
metabolic disease, and skin f lap surgery under diabetic condition represents a number of difficult pathophysiological problems. Diabetes aggravates skin flap necrosis,2 and potential new therapeutics for improving flap survival/suppressing necrosis are being investigated. Many studies have focused on antioxidants for protection against flap necrosis and other related complications.3Y5 Chlorogenic acid (CGA) is a phenolic compound that occurs naturally in plants, fruits, and vegetables.6 Chlorogenic acid has antioxidant, free radical scavenging, anti-inflammatory, analgesic, antiulcerogenic, and hypoglycemic effects.7Y11 The successful therapeutic potential of topical CGA treatment on an excisional wound model has been reported recently.12 In our previous study, long-term systemic administration of CGA enhanced flap survival in a random-pattern dorsal skin flap model in rats.13 The epigastric artery (EA)Ybased experimental f lap model is a useful strategy for understanding the effects of local treatments of drugs.14Y16 Furthermore, injections into the EA during f lap surgery are performed for local activity of various therapeutic agents.17Y19 In the current study, we aimed to investigate the effect of intra-arterial CGA in an EA-based abdominal skin f lap model in nondiabetic and diabetic rats.
(Ann Plast Surg 2014;00: 00Y00)
MATERIALS AND METHODS Animals
S
kin f laps are commonly used in the repair and reconstruction of local tissue loss and defects after tumor surgery and trauma. Flap necrosis is frequently observed in f lap tissues in the postoperative period and is an unwanted effect of healing.1 Diabetes mellitus is a
Received March 8, 2014, and accepted for publication, after revision, June 6, 2014. From the *Experimental Animals Breeding and Research Center, Faculty of Medicine, †Department of Physiology, Faculty of Medicine, ‡Department of Pharmacology, Faculty of Medicine, §Department of Pathology, Faculty of Veterinary Medicine, and ||Department of Biology, Faculty of Science and Arts, Uludag University, Bursa, Turkey. Conflicts of interest and sources of funding: This research was funded by a grant from Uludag University (Grant no: KUAP [T] 2012/37) to Dr Deniz Bagdas. All authors of this research article have directly participated in the planning, execution, or analysis of this study. The authors’ responsibilities were as follows: Deniz Bagdas performed research and was granted funds by Uludag University. Deniz Bagdas and Betul Cam Etoz performed in vivo study. Deniz Bagdas and Zulfiye Gul performed biochemical assays. Sevda Inan Ozturkoglu and Musa Ozgur Ozyigit performed histopathological analyses. Deniz Bagdas and Nilufer Cinkilic analyzed the data and wrote the manuscript. Naciye Isbil Buyukcoskun and Kasim Ozluk contributed to scientific work and revised the manuscript. Mine Sibel Gurun contributed to scientific work, contributed to the study design, and revised the manuscript. Reprints: Deniz Bagdas, DVM, PhD, Experimental Animals Breeding and Research Center, Faculty of Medicine, Uludag University, Bursa 16059, Turkey. E-mail: [email protected]; Betul Cam Etoz, MD, Department of Physiology, Faculty of Medicine, Uludag University, Bursa 16059, Turkey. E-mail: [email protected]; [email protected]. Copyright * 2014 by Lippincott Williams & Wilkins ISSN: 0148-7043/14/0000-0000 DOI: 10.1097/SAP.0000000000000313
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The study was performed on 48 Wistar albino rats (supplied by the Experimental Animals Breeding and Research Center, Uludag University). The animals were housed in conventional cages with 1 rat per cage in a temperature- and humidity-controlled room (21-C T 2-C, 50% T 5%) on a 12-hour light/dark cycle. The rats were provided free access to water and food. The study was approved by the Local Ethics Committee for Animal Experiments, Uludag University (date of approval, June 5, 2012; Registration No: 2012-07/05).
Study Plan The rats were firstly divided into 2 equally sized groups: nondiabetic and diabetic groups. Then, 4 subgroups were created for each group: vehicle as well as 0.2 mg/0.5 mL, 1 mg/0.5 mL, and 5 mg/ 0.5 mL CGA treatments. Phosphate buffered saline (PBS), the vehicle of CGA, was administered to the control groups. Chlorogenic acid (Acros Organics, Geel, Belgium) was dissolved in 10 mM of PBS (pH 7.4) at 37-C in a dark room and stored in darkness until use. The dose of CGA was selected from preliminary experiments for this study (data not shown). Drugs were injected only once shortly after flap surgery.
Induction of Diabetes The blood glucose levels were assessed in a drop of blood obtained from the tail vein using a glucose meter (One Touch Select; LifeScan, Milpitas, Calif ). Experimental diabetes mellitus was induced by a single 45-mg/kg intraperitoneal injection of streptozotocin (STZ) (Sigma Chemical Co, St Louis, Mo).2 After the induction of diabetes, the rats received a solution of 6% sucrose in their drinking water for 24 hours. The nondiabetic rats were injected with citrate buffer (J.T. Baker, Center Valley, Pa) as the vehicle of STZ in an equivalent www.annalsplasticsurgery.com
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volume. Four days later, blood glucose assessments were repeated, and the rats with fasting blood glucose levels of more than 300 mg/dL were considered to be diabetic. The diabetic rats were used 6 weeks later, and the diabetic status was controlled on surgery and postoperative day 7.
Flap Surgery and Evaluation of Flap Survival All the surgical procedures were performed under 2.5% to 3.5% sevoflurane inhaled anesthesia. The surgical area was shaved with an electric razor and manually depilated (Veet hair removal cream) under light anesthesia 1 day before the operation. On the surgery day, the surgical area was covered with sterile drapes after iodine antisepsis. Abdominal skin flap was elevated as described previously.17 The f lap area 7 4 cm in size was drawn with a pen on the abdominal wall. A flap 2-cm wide that crossed the midline and the chest bone was taken as midline reference to make it standardized. The flap tissue was removed on the basis of the EA (Fig. 1). The f lap was resutured to the surgical bed with 4-0 silk sutures. The femoral artery branches were dissected for intra-arterial local injections. The drug injections were performed with a handmade 30-gauge microcatheter injector to the femoral artery, after 2 microvascular clamps were placed to the proximal femoral artery section and the distal area after the EA branching,
FIGURE 1. Design of experimental EA-based abdominal skin f lap model and intra-arterial injection. Each f lap was created 7 4 cm based on the right EA pedicle. The femoral artery branches were dissected for intra-arterial local injections. The drug injections were performed with a handmade 30-gauge microcatheter injector to the femoral artery, after 2 microvascular clamps were placed to the proximal femoral artery section and the distal area after the EA branching, leaving the EA as the single artery feeding the f lap during the injections. The microcatheter was removed, and the microvascular clamps were opened, and reperfusion was provided 10 minutes after the injections. 2
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leaving the EA as the single artery feeding the flap during the injections, as shown in Figure 1. The microcatheter was removed, and the microvascular clamps were opened, and reperfusion was provided 10 minutes after the injections. The procedure was concluded by also suturing the dissected skin. The rats were maintained under normal conditions for 7 days in their own cages. On postoperative day 7, the rats were killed, and an image of the last condition of the f lap was obtained with a digital camera (Nikon CoolPIX L25; Nikon Corporation, Tokyo, Japan) from a distance of 20 cm. The necrotic and viable areas of the total f laps were traced on clear transparent sheets, and the sheets were scanned as digital images. The images were evaluated blindly using Image J software (version 1.44p; National Institutes of Health, Bethesda, Md) to calculate the percentage of the survival area.17 The total size of each f lap tissue and the survival area was measured separately to determine the ratio of f lap survival area to the total f lap tissue area, and the results were presented as the percentage of the total f lap area (area % = [survival area/f lap area] 100).
Biochemical Assays for Oxidative Stress Markers Biochemical analyses were performed as described in our previous report (Bagdas et al,13 2014). Flap tissue samples were rapidly collected from the distal flap region (2 cm caudal to the distal margin) after the rats were killed. The samples were washed well in cold 0.9% NaCl and were immediately frozen at j20-C until use. On the assessment day, the tissues were cut into pieces on ice and homogenized by a Heidolph DIAX 900 homogenizer in a 2-mL volume of ice-cold 10% trichloroacetic acid (Merck Chemicals, Darmstadt, Germany). The samples were vortexed for 10 seconds and centrifuged (4000 rpm, 15 minutes, 4-C). The supernatants were used for analysis. Furthermore, the tissue protein levels were determined in 50 KL of homogenate according to the method of Lowry et al.20 Malondialdehyde (MDA) and reduced glutathione (GSH) were measured spectrophotometrically (Mannheim Boehringer Photometer 4010). The MDA levels of the flap tissue were determined by the thiobarbituric acid method21 by adding 1 mL of 0.67% thiobarbituric acid (Sigma Chemical Co, St Louis, Mo) solution to 0.5 mL of the supernatant in a glass tube and holding the tube at 100-C for 60 minutes. After cooling the tubes, the absorbance of the supernatant was read at 546 nm. The results were expressed as picomolar per milligram protein, and 1,1,3,3-Tetramethoxypropane (Sigma Chemical Co, St Louis, Mo) was used as the MDA standard. The GSH levels were measured according to the Ellman method22 by adding 2 mL of 0.3 M of Na2HPO4 I 2H2O (BioShop, Burlington, Ontario, Canada) and 0.2 mL of 0.4 mg/mL of 5,5¶dithiobis-2-nitrobenzoic acid (Sigma Chemical Co, St Louis, Mo) in 1% sodium citrate to 0.5 mL of the supernatant. After vortexing, the absorbance was read at 405 nm. The results were expressed as micromolar per milligram protein. Superoxide dismutase (SOD) activity was assessed using a commercial enzyme-linked immunosorbent assay kit (Sigma Chemical Co, St Louis, Mo). The principal of the assay was based on the production of O2 anions by the xanthine/xanthine oxidase system and the reduction of nitroblue tetrazolium.23 The results were expressed as units per milligram protein.
Histopathological Examination The distal region of the flaps (1 cm caudal to the distal margin) was used for the histopathological examination. The flap tissue samples from each group were collected and fixed in 10% buffered formalin solution until the tissues hardened. The tissue samples were embedded in paraffin wax, cut into 5-KmYthick tissue sections, mounted on slides, and stained with hematoxylin-eosin. Each slide was examined quantitatively under a light microscope, as described in our previous study, graded as (0) absent, (1) slight, (2) moderate, (3) * 2014 Lippincott Williams & Wilkins
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Chlorogenic Acid in Skin Flap Survival
FIGURE 2. Photographic presentations of flaps on day 7. Nondiabetic and diabetic rats received 0.2 mg/0.5 mL, 1 mg/0.5 mL, or 5 mg/0.5 mL of CGA or PBS as vehicle.
marked, or (4) extensive for capillary density, edema, necrosis, and proliferation of fibroblasts on 200 magnification, and the mean number of findings was noted.13
Statistical Analysis Data are expressed as mean T SEM in all cases. Each group contained 5 to 6 rats. Data were analyzed using 1- or 2-way analysis of variance followed by the Student-Newman-Keuls test. A P value of less than 0.05 was considered significant.
groups, but the highest dose of CGA did not show a better result from the median dose. Diabetes caused markedly higher MDA levels as well as lower GSH and SOD levels in flap tissues in the vehicle-treated group when compared with the nondiabetic vehicle-treated rats. Chlorogenic acid attenuated the oxidative stress markers with increasing doses. Chlorogenic acid significantly decreased the MDA levels in 1- and 5-mg doses in the nondiabetic rats as well as in 5-mg dose in the diabetic rats.
RESULTS The blood glucose levels were significantly higher in the diabetic rats than the nondiabetic rats after STZ treatment on surgery day (431.5 T 18.6 and 84.8 T 1.5, respectively; P G 0.001). Chlorogenic acid treatments did not affect the blood glucose level in the diabetic rats (data not shown). The diabetic rats had significantly lower body weight than the nondiabetic rats (289.6 T 7.3 and 326.5 T 7.8 on surgery day, respectively; P G 0.001). There was no significant body weight gain during the course of the study by the treatments (data not shown). Except 1 animal from the vehicle-treated diabetic group, all animals survived on postoperative day 7. Necrotic areas of the f lap were dark in color with a rough texture, whereas the f lap survival area had a normal color and normal texture (Fig. 2). The f lap survival ratio is shown in Figure 3. In the nondiabetic rats, f lap survival ratios were 55.9 T 5.4 in the vehicle group as well as 81.0 T 1.6, 91.3 T 1.7, and 77.4 T 5.0 in the 0.2, 1, and 5 mg CGA-treated groups, respectively. Chlorogenic acid with doses of 0.2 and 1 mg enhanced f lap survival in a dose-dependent manner significantly. Interestingly, the highest dose of CGA in this study showed a significantly better f lap survival than in the vehicle-treated rats, but it was lower than expected. In the diabetic rats, f lap survival ratios were 46.0 T 6.1 in the vehicle group as well as 73.9 T 5.8, 87.8 T 6.0, and 69.8 T 5.4 in the 0.2, 1, and 5 mg CGA-treated groups, respectively. All diabetic rats showed considerably lower f lap survival ratios when compared with the nondiabetic controls, but the differences were not significant. Chlorogenic acid significantly increased f lap survival in all 3 * 2014 Lippincott Williams & Wilkins
FIGURE 3. The mean f lap survival rate (percentage). Nondiabetic and diabetic rats received 0.2 mg/0.5 mL, 1 mg/ 0.5 mL, or 5 mg/0.5 mL of CGA or PBS as vehicle. Data are expressed as the mean T SEM of 5 to 6 animals for each group. Statistical analysis was performed using 2-way analysis of variance followed by Student-Newman-Keuls test. *P G 0.05 and ***P G 0.001 are significantly different from the vehicle-treated nondiabetic rats. #P G 0.05, ##P G 0.01, and ###P G 0.001 are significantly different from the vehicle-treated diabetic rats. www.annalsplasticsurgery.com
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TABLE 1. The Oxidative Stress Markers of Flap Tissues
MDA, pM/mg protein GSH, KM/mg protein SOD, U/mg protein
Nondiabetic Rats
Diabetic Rats
Dose of CGA (mg) in 0.5-mL PBS
Dose of CGA (mg) in 0.5-mL PBS
0
0.2
1
5
0
0.2
1
5
381 T 83 16.9 T 5.1 7.2 T 1.3
361 T 126 15.6 T 34 13.6 T 4.4
140 T 23* 15.9 T 2.8 9.9 T 2.7
150 T 25* 14.5 T 5. 5.8 T 2.3
599 T 116† 7.2 T 1.5† 4.4 T 1.5†
463 T 121 10.9 T 1.3 9.8 T 3.0
345 T 76 16.4 T 3.3* 22.4 T 5.8*
253 T 77* 9.1 T 0.6 9.2 T 3.0
*p G 0.05 vs. its control †p 9 0.05 vs. nondiabetic control.
Glutathione and SOD levels of flap tissues slightly increased with CGA treatments. Significant differences were found in 1-mg CGA therapy in GSH and SOD levels when compared with the vehicle-treated diabetic rats (Table 1). The histopathological results showed lesser necrosis in the CGA-treated rats. In addition, CGA treatment resulted in better vascularization by increasing capillary density in both nondiabetic and diabetic rats. There were no clear differences between the groups about edema and fibroblast proliferation (Table 2).
DISCUSSION The present study shows for the first time that intra-arterial local administration of CGA promotes significant healing in an experimental abdominal skin f lap model in nondiabetic and diabetic rats. Chlorogenic acid significantly decreased the MDA levels and increased the GSH and SOD levels. Histopathological examinations revealed increased capillary density and decreased necrosis. Chlorogenic acid did not show antidiabetic effects by 1-time intraarterial administration. Chlorogenic acid has been reported to exhibit potent antioxidant and anti-inf lammatory activities.6,9,13 Chlorogenic acid may have particularly beneficial effects on healing as a result of its properties. The wound healing effect of topical CGA treatment on an excisional wound model was recently reported. Topical CGA cream accelerated wound healing by its antioxidant activity on skin.12 In addition, our unpublished investigations on long-term systemic CGA administrations on excisional wounds in diabetic rats also showed that CGA has strong wound healing activity in diabetic animals. In our previous study, we also observed that long-term intraperitoneal CGA treatments have a beneficial role in f lap viability. The systemic administration of CGA enhanced regional perfusion and promoted significant healing activity by its antioxidant, anti-inf lammatory, neovascularization, and free radical scavenger properties on f lap survival in an experimental random-pattern dorsal skin f lap model in rats.13 The results in the current study are consistent with our previous and wound healing studies. In addition, the relative effectiveness of different administration routes may be summarized as that a single intra-arterial injection of 1-mg dose of CGA is more effective in reducing skin f lap necrosis. In addition, CGA has great potential
clinical utility because it has been presented traditionally taken orally. In particular, supplementary CGA may have a beneficial role in the postoperative period. In a study, CGA was infused in a dosage of 5 mg/kg body weight per day for 3 weeks via intravenous infusion to insulin-resistant Zucker rats. Chlorogenic acid did not lower the fasting blood glucose levels of rats, but CGA showed some antihyperlipidemic effects.24 In our study, we used intra-arterial administration for feeding the flap area. Chlorogenic acid showed some side effects with 5 mg and higher doses (data not shown). The increasing doses of CGA caused similar f lap necrosis with vehicle-treated rats. The results indicate that direct intra-arterial administration of CGA in higher doses than 5 mg/0.5 mL may be toxic to skin flaps. Additional studies are needed to clarify the reason of the side effects in higher doses, but we suggest that the acidic activity may change the healing effects. Diabetes has adverse effects on f lap survival. The most common complication of diabetes in surgical f laps is increased partialf lap necrosis.2 In the present study, diabetes slightly increased the necrosis, but it was not significantly different from the necrosis in the nondiabetic controls. On the other hand, the results indicated significant decay in biochemical examination of diabetic f lap tissues. In the current study, hyperglycemia was provided by a 1-time dose of STZ, which is highly toxic to pancreatic islet cells and a welldocumented method for mimicking type 1 diabetes in animals. The exact effect of diabetic state on skin systems is still being investigated experimentally. This method may partly differ from clinical diabetes mellitus in chronicity, effect on the immune system, vascular disease, and others, which may state the mild differences in f lap survival seen between the diabetic and nondiabetic animals in the present study. Furthermore, CGA has been reported to modulate blood glucose levels and exhibits hypoglycemic activity with long-term administrations. It has been proven to show the hypoglycemic effects in diabetes by directly inhibiting the activity of glucose-6-phosphatase, which is involved in glucose metabolism.11 In this study, we did not observe any difference in the blood glucose level of the diabetic rats with 1-time intra-arterial treatment. On the other hand, CGA has been hypothesized for increasing the glucagon-like peptide-1 (GLP-1) hormone production.25 Glucagon-like peptide-1 also has various beneficial effects on blood sugar levels, and GLP-1 has been found
TABLE 2. Semiquantitative Histopathological Findings of Flap Tissues Nondiabetic Rats
Diabetic Rat
Dose of CGA (mg) in 0.5-mL PBS
Capillary density Edema Necrosis Fibroblast proliferation
4
Dose of CGA (mg) in 0.5-mL PBS
0
0.2
1
5
0
0.2
1
5
1.4 2.7 2.5 2.3
2.5 2.5 1.2 2.2
2.8 2.6 0.8 2
2.5 2.5 0.3 2.5
1.2 2.4 3 2.2
2.3 2.8 1.2 2.7
2.6 2.7 1.2 2.2
1.8 2.4 0.6 1.6
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by our coauthors17 to improve abdominal f lap survival by intraarterial injections into the EA. Chlorogenic acid may relate to the improving healing activity by GLP-1 production. Further studies are needed to understand the relation of this hypothesis, but we suggest that CGA is a superior antioxidant compared with other supraoxide radical scavengers by its possible precursor activity for caffeic acid and possible beneficial activities on GLP-1 production also. Ischemic injury participates in the pathophysiology of many surgical conditions, and oxidative and inf lammatory reactions occur in the ischemic tissue as a result of the injury response. Free radicals, released by endothelial and inf lammatory cells such as neutrophils and macrophages, are thought to be involved in ischemic damage. The generation of reactive oxygen radical species (ROS) in skin tissue is one of the mechanisms for skin f lap necrosis.5,26,27 Reactive oxygen radical species generation is the major cause of lipid peroxidation, which is aggravated by the presence of diabetes. A variety of antioxidant defense systems have been developed by the organism as a protective mechanism against ROS. The endogenous antioxidant systems for protection from ROS include SOD, GSH, and catalase.5,27 Chlorogenic acid may also act as free radical scavengers.11,13 Chlorogenic acid has been shown to suppress surgical oxidative stress, as indicated by decreasing MDA (marker of lipid peroxidation) with increasing GSH and SOD levels in skin tissues.12,13 Chlorogenic acid is formed by the esterification of caffeic and quinic acid.6 Caffeic acid phenethyl ester was shown to exhibit beneficial effects on dorsal skin f lap survival and protective effects against ischemia/reperfusion injury by surgical skin f lap. Caffeic acid phenethyl ester exhibited healing effects on f lap survival by decreasing the MDA and nitric oxide levels and increasing the GSH and SOD levels in f lap tissue.3,4 In addition, CGA showed enhanced f lap survival by attenuating oxidative stress markers in our previous study.13 In accordance with these results, higher doses of CGA decreased MDA levels and increased the GSH and SOD levels of the f lap tissues in the current study. We suggest that CGA may improve f lap survival because of its antioxidant properties. We also propose to further investigate the EA route for comparing CGA and its metabolites. In summary, CGA enhanced abdominal skin f lap survival based on EA in nondiabetic and diabetic rats. Although there are many limitations in this study, the beneficial effects of CGA in improving f lap survival may be explained by its antioxidant activity. ACKNOWLEDGMENT The authors thank Y. Bagdas for English editing of the manuscript.
REFERENCES 1. Myers B. Understanding flap necrosis. Plast Reconstr Surg. 1986;78:813Y814. 2. Isken T, Ozgentas HE, Gulkesen KH, et al. A random-pattern skin-flap model in streptozotocin diabetic rats. Ann Plast Surg. 2006;57:323Y329. 3. Aydogan H, Gurlek A, Parlakpinar H, et al. Beneficial effects of caffeic acid phenethyl ester (CAPE) on the ischaemia-reperfusion injury in rat skin flaps. J Plast Reconstr Aesthet Surg. 2007;60:563Y568. 4. Bilen BT, Kilinc¸ H, Alaybeyoglu N, et al. Effect of caffeic acid phenethyl ester on survival of axial pattern flaps in rats with ischaemia-reperfusion injuries. Scand J Plast Reconstr Surg Hand Surg. 2006;40:73Y78.
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Chlorogenic Acid in Skin Flap Survival
5. Ozyazgan I, Tuncer A, Yazici C, et al. Reactive oxygen species in experimental ischemic flow-through venous flaps and effects of antioxidants on reactive oxygen species and flap survival. Ann Plast Surg. 2007;58:661Y666. 6. Clifford MN. Chlorogenic acids and other cinnamatesVnature, occurrence and dietary burden. J Sci Food Agric. 1999;79:362Y372. 7. Bagdas D, Cinkilic N, Ozboluk HY, et al. Antihyperalgesic activity of chlorogenic acid in experimental neuropathic pain. J Nat Med. 2013;67: 698Y704. 8. Cinkilic N, Cetintas SK, Zorlu T, et al. Radioprotection by two phenolic compounds: chlorogenic and quinic acid, on x-ray induced DNA damage in human blood lymphocytes in vitro. Food Chem Toxicol. 2013;53:359Y363. 9. dos Santos MD, Almeida MC, Lopes NP, et al. Evaluation of the antiinflammatory, analgesic and antipyretic activities of the natural polyphenol chlorogenic acid. Biol Pharm Bull. 2006;29:2236Y2240. 10. Shimoyama AT, Santin JR, Machado ID, et al. Antiulcerogenic activity of chlorogenic acid in different models of gastric ulcer. Naunyn Schmiedebergs Arch Pharmacol. 2013;386:5Y14. 11. Karthikesan K, Pari L, Menon VP. Combined treatment of tetrahydrocurcumin and chlorogenic acid exerts potential antihyperglycemic effect on streptozotocin-nicotinamide-induced diabetic rats. Gen Physiol Biophys. 2010; 29:23Y30. 12. Chen WC, Liou SS, Tzeng TF, et al. Effect of topical application of chlorogenic acid on excision wound healing in rats. Planta Med. 2013;79:616Y621. 13. Bagdas D, Cam-Etoz B, Inan-Ozturkoglu S, et al. Effects of systemic chlorogenic acid on random-pattern dorsal skin flap survival in diabetic rats. Biol Pharm Bull. 2014;37:361Y70. 14. Gribbe O, Gustafsson LE, Wiklund NP. Transdermally administered nitric oxide by application of acidified nitrite increases blood flow in rat epigastric island skin flaps. Eur J Pharmacol. 2008;578:51Y56. 15. Fromes Y, Liu JM, Kovacevic M, et al. The tetrapeptide acetyl-serine-aspartyllysine-proline improves skin flap survival and accelerates wound healing. Wound Repair Regen. 2006;14:306Y312. 16. Yamashita S, Sugiyama N, Hasegawa K, et al. A novel model for supermicrosurgery training: the superficial inferior epigastric artery flap in rats. J Reconstr Microsurg. 2008;24:537Y543. 17. Cam Etoz B, Isbil Buyukcoskun N, Etoz A, et al. Preventing flap necrosis with adrenomedullin and glucagon like peptide-1. Wounds. 2012;24:29Y35. 18. Komorowska-Timek E, Timek TA, Brevetti LS, et al. The effect of single administration of vascular endothelial growth factor or L-arginine on necrosis and vasculature of the epigastric flap in the rat model. Br J Plast Surg. 2004;57:317Y325. 19. Scalise A, Tucci MG, Lucarini G, et al. Local rh-VEGF administration enhances skin flap survival more than other types of rh-VEGF administration: a clinical, morphological and immunohistochemical study. Exp Dermatol. 2004; 13:682Y690. 20. Lowry OH, Rosebrough NJ, Farr AL, et al. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951;193:265Y275. 21. Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem. 1979;95:351Y358. 22. Ellman GL. Tissue sulfhydryl groups. Arch Biochem Biophys. 1959;82:70Y77. 23. Sun Y, Oberley LW, Li Y. A simple method for clinical assay of superoxide dismutase. Clin Chem. 1988;34:497Y500. 24. Rodriguez de Sotillo DV, Hadley M. Chlorogenic acid modifies plasma and liver concentrations of: cholesterol, triacylglycerol, and minerals in (fa/fa) Zucker rats. J Nutr Biochem. 2002;13:717Y726. 25. McCarty MF. A chlorogenic acid-induced increase in GLP-1 production may mediate the impact of heavy coffee consumption on diabetes risk. Med Hypotheses. 2005;64:848Y853. 26. Carden DL, Granger DN. Pathophysiology of ischaemia-reperfusion injury. J Pathol. 2000;190:255Y266. 27. Khalil AA, Aziz FA, Hall JC. Reperfusion injury. Plast Reconstr Surg. 2006; 117:1024Y1033.
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Chlorogenic Acid Enhances Abdominal Skin Flap Survival Based on Epigastric Artery in Nondiabetic and Diabetic Rats Deniz Bagdas, DVM, PhD,* Betul Cam Etoz, MD, PhD,Þ Zulfiye Gul, MS,þ Musa Ozgur Ozyigit, DVM, PhD,§ Nilufer Cinkilic, PhD,|| Sevda Inan, DVM, MS,§ Naciye Isbil Buyukcoskun, PhD,Þ Kasim Ozluk, PhD,Þ and Mine Sibel Gurun, MD, PhDþ Abstract: Previous studies showed that chlorogenic acid (CGA) accelerates wound healing via its antioxidant activity. We aimed to investigate the effect of CGA in an experimental epigastric abdominal skin f lap model in nondiabetic and diabetic rats. Rats were firstly divided into 2 groups: nondiabetic and diabetic. Diabetes was induced by streptozotocin. Then, 4 subgroups were created for each group: vehicle as well as 0.2 mg/0.5 mL, 1 mg/0.5 mL, and 5 mg/0.5 mL CGA treatments. Right epigastric arteryYbased abdominal skin f laps were elevated and sutured back into their original position. Chlorogenic acid or vehicle was injected once into the femoral arteries by leaving the epigastric artery as the single artery feeding the f laps during the injection. On postoperative day 7, f lap survivals were evaluated, and the rats were killed. Distal f lap tissues were collected for histopathological and biochemical assays. Chlorogenic acid showed greater f lap survival in both nondiabetic and diabetic rats. Capillary density was increased, and necrosis was reduced in the CGA-treated rats. Chlorogenic acid decreased malondialdehyde levels as well as increased reduced glutathione and superoxide dismutase levels in the f lap tissues. This study showed that CGA significantly improved f lap survival by its antioxidant activities with intra-arterial local injections. Key Words: antioxidant, chlorogenic acid, diabetes, epigastric artery, flap survival
metabolic disease, and skin f lap surgery under diabetic condition represents a number of difficult pathophysiological problems. Diabetes aggravates skin flap necrosis,2 and potential new therapeutics for improving flap survival/suppressing necrosis are being investigated. Many studies have focused on antioxidants for protection against flap necrosis and other related complications.3Y5 Chlorogenic acid (CGA) is a phenolic compound that occurs naturally in plants, fruits, and vegetables.6 Chlorogenic acid has antioxidant, free radical scavenging, anti-inflammatory, analgesic, antiulcerogenic, and hypoglycemic effects.7Y11 The successful therapeutic potential of topical CGA treatment on an excisional wound model has been reported recently.12 In our previous study, long-term systemic administration of CGA enhanced flap survival in a random-pattern dorsal skin flap model in rats.13 The epigastric artery (EA)Ybased experimental f lap model is a useful strategy for understanding the effects of local treatments of drugs.14Y16 Furthermore, injections into the EA during f lap surgery are performed for local activity of various therapeutic agents.17Y19 In the current study, we aimed to investigate the effect of intra-arterial CGA in an EA-based abdominal skin f lap model in nondiabetic and diabetic rats.
(Ann Plast Surg 2014;00: 00Y00)
MATERIALS AND METHODS Animals
S
kin f laps are commonly used in the repair and reconstruction of local tissue loss and defects after tumor surgery and trauma. Flap necrosis is frequently observed in f lap tissues in the postoperative period and is an unwanted effect of healing.1 Diabetes mellitus is a
Received March 8, 2014, and accepted for publication, after revision, June 6, 2014. From the *Experimental Animals Breeding and Research Center, Faculty of Medicine, †Department of Physiology, Faculty of Medicine, ‡Department of Pharmacology, Faculty of Medicine, §Department of Pathology, Faculty of Veterinary Medicine, and ||Department of Biology, Faculty of Science and Arts, Uludag University, Bursa, Turkey. Conflicts of interest and sources of funding: This research was funded by a grant from Uludag University (Grant no: KUAP [T] 2012/37) to Dr Deniz Bagdas. All authors of this research article have directly participated in the planning, execution, or analysis of this study. The authors’ responsibilities were as follows: Deniz Bagdas performed research and was granted funds by Uludag University. Deniz Bagdas and Betul Cam Etoz performed in vivo study. Deniz Bagdas and Zulfiye Gul performed biochemical assays. Sevda Inan Ozturkoglu and Musa Ozgur Ozyigit performed histopathological analyses. Deniz Bagdas and Nilufer Cinkilic analyzed the data and wrote the manuscript. Naciye Isbil Buyukcoskun and Kasim Ozluk contributed to scientific work and revised the manuscript. Mine Sibel Gurun contributed to scientific work, contributed to the study design, and revised the manuscript. Reprints: Deniz Bagdas, DVM, PhD, Experimental Animals Breeding and Research Center, Faculty of Medicine, Uludag University, Bursa 16059, Turkey. E-mail: [email protected]; Betul Cam Etoz, MD, Department of Physiology, Faculty of Medicine, Uludag University, Bursa 16059, Turkey. E-mail: [email protected]; [email protected]. Copyright * 2014 by Lippincott Williams & Wilkins ISSN: 0148-7043/14/0000-0000 DOI: 10.1097/SAP.0000000000000313
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The study was performed on 48 Wistar albino rats (supplied by the Experimental Animals Breeding and Research Center, Uludag University). The animals were housed in conventional cages with 1 rat per cage in a temperature- and humidity-controlled room (21-C T 2-C, 50% T 5%) on a 12-hour light/dark cycle. The rats were provided free access to water and food. The study was approved by the Local Ethics Committee for Animal Experiments, Uludag University (date of approval, June 5, 2012; Registration No: 2012-07/05).
Study Plan The rats were firstly divided into 2 equally sized groups: nondiabetic and diabetic groups. Then, 4 subgroups were created for each group: vehicle as well as 0.2 mg/0.5 mL, 1 mg/0.5 mL, and 5 mg/ 0.5 mL CGA treatments. Phosphate buffered saline (PBS), the vehicle of CGA, was administered to the control groups. Chlorogenic acid (Acros Organics, Geel, Belgium) was dissolved in 10 mM of PBS (pH 7.4) at 37-C in a dark room and stored in darkness until use. The dose of CGA was selected from preliminary experiments for this study (data not shown). Drugs were injected only once shortly after flap surgery.
Induction of Diabetes The blood glucose levels were assessed in a drop of blood obtained from the tail vein using a glucose meter (One Touch Select; LifeScan, Milpitas, Calif ). Experimental diabetes mellitus was induced by a single 45-mg/kg intraperitoneal injection of streptozotocin (STZ) (Sigma Chemical Co, St Louis, Mo).2 After the induction of diabetes, the rats received a solution of 6% sucrose in their drinking water for 24 hours. The nondiabetic rats were injected with citrate buffer (J.T. Baker, Center Valley, Pa) as the vehicle of STZ in an equivalent www.annalsplasticsurgery.com
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volume. Four days later, blood glucose assessments were repeated, and the rats with fasting blood glucose levels of more than 300 mg/dL were considered to be diabetic. The diabetic rats were used 6 weeks later, and the diabetic status was controlled on surgery and postoperative day 7.
Flap Surgery and Evaluation of Flap Survival All the surgical procedures were performed under 2.5% to 3.5% sevoflurane inhaled anesthesia. The surgical area was shaved with an electric razor and manually depilated (Veet hair removal cream) under light anesthesia 1 day before the operation. On the surgery day, the surgical area was covered with sterile drapes after iodine antisepsis. Abdominal skin flap was elevated as described previously.17 The f lap area 7 4 cm in size was drawn with a pen on the abdominal wall. A flap 2-cm wide that crossed the midline and the chest bone was taken as midline reference to make it standardized. The flap tissue was removed on the basis of the EA (Fig. 1). The f lap was resutured to the surgical bed with 4-0 silk sutures. The femoral artery branches were dissected for intra-arterial local injections. The drug injections were performed with a handmade 30-gauge microcatheter injector to the femoral artery, after 2 microvascular clamps were placed to the proximal femoral artery section and the distal area after the EA branching,
FIGURE 1. Design of experimental EA-based abdominal skin f lap model and intra-arterial injection. Each f lap was created 7 4 cm based on the right EA pedicle. The femoral artery branches were dissected for intra-arterial local injections. The drug injections were performed with a handmade 30-gauge microcatheter injector to the femoral artery, after 2 microvascular clamps were placed to the proximal femoral artery section and the distal area after the EA branching, leaving the EA as the single artery feeding the f lap during the injections. The microcatheter was removed, and the microvascular clamps were opened, and reperfusion was provided 10 minutes after the injections. 2
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leaving the EA as the single artery feeding the flap during the injections, as shown in Figure 1. The microcatheter was removed, and the microvascular clamps were opened, and reperfusion was provided 10 minutes after the injections. The procedure was concluded by also suturing the dissected skin. The rats were maintained under normal conditions for 7 days in their own cages. On postoperative day 7, the rats were killed, and an image of the last condition of the f lap was obtained with a digital camera (Nikon CoolPIX L25; Nikon Corporation, Tokyo, Japan) from a distance of 20 cm. The necrotic and viable areas of the total f laps were traced on clear transparent sheets, and the sheets were scanned as digital images. The images were evaluated blindly using Image J software (version 1.44p; National Institutes of Health, Bethesda, Md) to calculate the percentage of the survival area.17 The total size of each f lap tissue and the survival area was measured separately to determine the ratio of f lap survival area to the total f lap tissue area, and the results were presented as the percentage of the total f lap area (area % = [survival area/f lap area] 100).
Biochemical Assays for Oxidative Stress Markers Biochemical analyses were performed as described in our previous report (Bagdas et al,13 2014). Flap tissue samples were rapidly collected from the distal flap region (2 cm caudal to the distal margin) after the rats were killed. The samples were washed well in cold 0.9% NaCl and were immediately frozen at j20-C until use. On the assessment day, the tissues were cut into pieces on ice and homogenized by a Heidolph DIAX 900 homogenizer in a 2-mL volume of ice-cold 10% trichloroacetic acid (Merck Chemicals, Darmstadt, Germany). The samples were vortexed for 10 seconds and centrifuged (4000 rpm, 15 minutes, 4-C). The supernatants were used for analysis. Furthermore, the tissue protein levels were determined in 50 KL of homogenate according to the method of Lowry et al.20 Malondialdehyde (MDA) and reduced glutathione (GSH) were measured spectrophotometrically (Mannheim Boehringer Photometer 4010). The MDA levels of the flap tissue were determined by the thiobarbituric acid method21 by adding 1 mL of 0.67% thiobarbituric acid (Sigma Chemical Co, St Louis, Mo) solution to 0.5 mL of the supernatant in a glass tube and holding the tube at 100-C for 60 minutes. After cooling the tubes, the absorbance of the supernatant was read at 546 nm. The results were expressed as picomolar per milligram protein, and 1,1,3,3-Tetramethoxypropane (Sigma Chemical Co, St Louis, Mo) was used as the MDA standard. The GSH levels were measured according to the Ellman method22 by adding 2 mL of 0.3 M of Na2HPO4 I 2H2O (BioShop, Burlington, Ontario, Canada) and 0.2 mL of 0.4 mg/mL of 5,5¶dithiobis-2-nitrobenzoic acid (Sigma Chemical Co, St Louis, Mo) in 1% sodium citrate to 0.5 mL of the supernatant. After vortexing, the absorbance was read at 405 nm. The results were expressed as micromolar per milligram protein. Superoxide dismutase (SOD) activity was assessed using a commercial enzyme-linked immunosorbent assay kit (Sigma Chemical Co, St Louis, Mo). The principal of the assay was based on the production of O2 anions by the xanthine/xanthine oxidase system and the reduction of nitroblue tetrazolium.23 The results were expressed as units per milligram protein.
Histopathological Examination The distal region of the flaps (1 cm caudal to the distal margin) was used for the histopathological examination. The flap tissue samples from each group were collected and fixed in 10% buffered formalin solution until the tissues hardened. The tissue samples were embedded in paraffin wax, cut into 5-KmYthick tissue sections, mounted on slides, and stained with hematoxylin-eosin. Each slide was examined quantitatively under a light microscope, as described in our previous study, graded as (0) absent, (1) slight, (2) moderate, (3) * 2014 Lippincott Williams & Wilkins
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Chlorogenic Acid in Skin Flap Survival
FIGURE 2. Photographic presentations of flaps on day 7. Nondiabetic and diabetic rats received 0.2 mg/0.5 mL, 1 mg/0.5 mL, or 5 mg/0.5 mL of CGA or PBS as vehicle.
marked, or (4) extensive for capillary density, edema, necrosis, and proliferation of fibroblasts on 200 magnification, and the mean number of findings was noted.13
Statistical Analysis Data are expressed as mean T SEM in all cases. Each group contained 5 to 6 rats. Data were analyzed using 1- or 2-way analysis of variance followed by the Student-Newman-Keuls test. A P value of less than 0.05 was considered significant.
groups, but the highest dose of CGA did not show a better result from the median dose. Diabetes caused markedly higher MDA levels as well as lower GSH and SOD levels in flap tissues in the vehicle-treated group when compared with the nondiabetic vehicle-treated rats. Chlorogenic acid attenuated the oxidative stress markers with increasing doses. Chlorogenic acid significantly decreased the MDA levels in 1- and 5-mg doses in the nondiabetic rats as well as in 5-mg dose in the diabetic rats.
RESULTS The blood glucose levels were significantly higher in the diabetic rats than the nondiabetic rats after STZ treatment on surgery day (431.5 T 18.6 and 84.8 T 1.5, respectively; P G 0.001). Chlorogenic acid treatments did not affect the blood glucose level in the diabetic rats (data not shown). The diabetic rats had significantly lower body weight than the nondiabetic rats (289.6 T 7.3 and 326.5 T 7.8 on surgery day, respectively; P G 0.001). There was no significant body weight gain during the course of the study by the treatments (data not shown). Except 1 animal from the vehicle-treated diabetic group, all animals survived on postoperative day 7. Necrotic areas of the f lap were dark in color with a rough texture, whereas the f lap survival area had a normal color and normal texture (Fig. 2). The f lap survival ratio is shown in Figure 3. In the nondiabetic rats, f lap survival ratios were 55.9 T 5.4 in the vehicle group as well as 81.0 T 1.6, 91.3 T 1.7, and 77.4 T 5.0 in the 0.2, 1, and 5 mg CGA-treated groups, respectively. Chlorogenic acid with doses of 0.2 and 1 mg enhanced f lap survival in a dose-dependent manner significantly. Interestingly, the highest dose of CGA in this study showed a significantly better f lap survival than in the vehicle-treated rats, but it was lower than expected. In the diabetic rats, f lap survival ratios were 46.0 T 6.1 in the vehicle group as well as 73.9 T 5.8, 87.8 T 6.0, and 69.8 T 5.4 in the 0.2, 1, and 5 mg CGA-treated groups, respectively. All diabetic rats showed considerably lower f lap survival ratios when compared with the nondiabetic controls, but the differences were not significant. Chlorogenic acid significantly increased f lap survival in all 3 * 2014 Lippincott Williams & Wilkins
FIGURE 3. The mean f lap survival rate (percentage). Nondiabetic and diabetic rats received 0.2 mg/0.5 mL, 1 mg/ 0.5 mL, or 5 mg/0.5 mL of CGA or PBS as vehicle. Data are expressed as the mean T SEM of 5 to 6 animals for each group. Statistical analysis was performed using 2-way analysis of variance followed by Student-Newman-Keuls test. *P G 0.05 and ***P G 0.001 are significantly different from the vehicle-treated nondiabetic rats. #P G 0.05, ##P G 0.01, and ###P G 0.001 are significantly different from the vehicle-treated diabetic rats. www.annalsplasticsurgery.com
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TABLE 1. The Oxidative Stress Markers of Flap Tissues
MDA, pM/mg protein GSH, KM/mg protein SOD, U/mg protein
Nondiabetic Rats
Diabetic Rats
Dose of CGA (mg) in 0.5-mL PBS
Dose of CGA (mg) in 0.5-mL PBS
0
0.2
1
5
0
0.2
1
5
381 T 83 16.9 T 5.1 7.2 T 1.3
361 T 126 15.6 T 34 13.6 T 4.4
140 T 23* 15.9 T 2.8 9.9 T 2.7
150 T 25* 14.5 T 5. 5.8 T 2.3
599 T 116† 7.2 T 1.5† 4.4 T 1.5†
463 T 121 10.9 T 1.3 9.8 T 3.0
345 T 76 16.4 T 3.3* 22.4 T 5.8*
253 T 77* 9.1 T 0.6 9.2 T 3.0
*p G 0.05 vs. its control †p 9 0.05 vs. nondiabetic control.
Glutathione and SOD levels of flap tissues slightly increased with CGA treatments. Significant differences were found in 1-mg CGA therapy in GSH and SOD levels when compared with the vehicle-treated diabetic rats (Table 1). The histopathological results showed lesser necrosis in the CGA-treated rats. In addition, CGA treatment resulted in better vascularization by increasing capillary density in both nondiabetic and diabetic rats. There were no clear differences between the groups about edema and fibroblast proliferation (Table 2).
DISCUSSION The present study shows for the first time that intra-arterial local administration of CGA promotes significant healing in an experimental abdominal skin f lap model in nondiabetic and diabetic rats. Chlorogenic acid significantly decreased the MDA levels and increased the GSH and SOD levels. Histopathological examinations revealed increased capillary density and decreased necrosis. Chlorogenic acid did not show antidiabetic effects by 1-time intraarterial administration. Chlorogenic acid has been reported to exhibit potent antioxidant and anti-inf lammatory activities.6,9,13 Chlorogenic acid may have particularly beneficial effects on healing as a result of its properties. The wound healing effect of topical CGA treatment on an excisional wound model was recently reported. Topical CGA cream accelerated wound healing by its antioxidant activity on skin.12 In addition, our unpublished investigations on long-term systemic CGA administrations on excisional wounds in diabetic rats also showed that CGA has strong wound healing activity in diabetic animals. In our previous study, we also observed that long-term intraperitoneal CGA treatments have a beneficial role in f lap viability. The systemic administration of CGA enhanced regional perfusion and promoted significant healing activity by its antioxidant, anti-inf lammatory, neovascularization, and free radical scavenger properties on f lap survival in an experimental random-pattern dorsal skin f lap model in rats.13 The results in the current study are consistent with our previous and wound healing studies. In addition, the relative effectiveness of different administration routes may be summarized as that a single intra-arterial injection of 1-mg dose of CGA is more effective in reducing skin f lap necrosis. In addition, CGA has great potential
clinical utility because it has been presented traditionally taken orally. In particular, supplementary CGA may have a beneficial role in the postoperative period. In a study, CGA was infused in a dosage of 5 mg/kg body weight per day for 3 weeks via intravenous infusion to insulin-resistant Zucker rats. Chlorogenic acid did not lower the fasting blood glucose levels of rats, but CGA showed some antihyperlipidemic effects.24 In our study, we used intra-arterial administration for feeding the flap area. Chlorogenic acid showed some side effects with 5 mg and higher doses (data not shown). The increasing doses of CGA caused similar f lap necrosis with vehicle-treated rats. The results indicate that direct intra-arterial administration of CGA in higher doses than 5 mg/0.5 mL may be toxic to skin flaps. Additional studies are needed to clarify the reason of the side effects in higher doses, but we suggest that the acidic activity may change the healing effects. Diabetes has adverse effects on f lap survival. The most common complication of diabetes in surgical f laps is increased partialf lap necrosis.2 In the present study, diabetes slightly increased the necrosis, but it was not significantly different from the necrosis in the nondiabetic controls. On the other hand, the results indicated significant decay in biochemical examination of diabetic f lap tissues. In the current study, hyperglycemia was provided by a 1-time dose of STZ, which is highly toxic to pancreatic islet cells and a welldocumented method for mimicking type 1 diabetes in animals. The exact effect of diabetic state on skin systems is still being investigated experimentally. This method may partly differ from clinical diabetes mellitus in chronicity, effect on the immune system, vascular disease, and others, which may state the mild differences in f lap survival seen between the diabetic and nondiabetic animals in the present study. Furthermore, CGA has been reported to modulate blood glucose levels and exhibits hypoglycemic activity with long-term administrations. It has been proven to show the hypoglycemic effects in diabetes by directly inhibiting the activity of glucose-6-phosphatase, which is involved in glucose metabolism.11 In this study, we did not observe any difference in the blood glucose level of the diabetic rats with 1-time intra-arterial treatment. On the other hand, CGA has been hypothesized for increasing the glucagon-like peptide-1 (GLP-1) hormone production.25 Glucagon-like peptide-1 also has various beneficial effects on blood sugar levels, and GLP-1 has been found
TABLE 2. Semiquantitative Histopathological Findings of Flap Tissues Nondiabetic Rats
Diabetic Rat
Dose of CGA (mg) in 0.5-mL PBS
Capillary density Edema Necrosis Fibroblast proliferation
4
Dose of CGA (mg) in 0.5-mL PBS
0
0.2
1
5
0
0.2
1
5
1.4 2.7 2.5 2.3
2.5 2.5 1.2 2.2
2.8 2.6 0.8 2
2.5 2.5 0.3 2.5
1.2 2.4 3 2.2
2.3 2.8 1.2 2.7
2.6 2.7 1.2 2.2
1.8 2.4 0.6 1.6
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by our coauthors17 to improve abdominal f lap survival by intraarterial injections into the EA. Chlorogenic acid may relate to the improving healing activity by GLP-1 production. Further studies are needed to understand the relation of this hypothesis, but we suggest that CGA is a superior antioxidant compared with other supraoxide radical scavengers by its possible precursor activity for caffeic acid and possible beneficial activities on GLP-1 production also. Ischemic injury participates in the pathophysiology of many surgical conditions, and oxidative and inf lammatory reactions occur in the ischemic tissue as a result of the injury response. Free radicals, released by endothelial and inf lammatory cells such as neutrophils and macrophages, are thought to be involved in ischemic damage. The generation of reactive oxygen radical species (ROS) in skin tissue is one of the mechanisms for skin f lap necrosis.5,26,27 Reactive oxygen radical species generation is the major cause of lipid peroxidation, which is aggravated by the presence of diabetes. A variety of antioxidant defense systems have been developed by the organism as a protective mechanism against ROS. The endogenous antioxidant systems for protection from ROS include SOD, GSH, and catalase.5,27 Chlorogenic acid may also act as free radical scavengers.11,13 Chlorogenic acid has been shown to suppress surgical oxidative stress, as indicated by decreasing MDA (marker of lipid peroxidation) with increasing GSH and SOD levels in skin tissues.12,13 Chlorogenic acid is formed by the esterification of caffeic and quinic acid.6 Caffeic acid phenethyl ester was shown to exhibit beneficial effects on dorsal skin f lap survival and protective effects against ischemia/reperfusion injury by surgical skin f lap. Caffeic acid phenethyl ester exhibited healing effects on f lap survival by decreasing the MDA and nitric oxide levels and increasing the GSH and SOD levels in f lap tissue.3,4 In addition, CGA showed enhanced f lap survival by attenuating oxidative stress markers in our previous study.13 In accordance with these results, higher doses of CGA decreased MDA levels and increased the GSH and SOD levels of the f lap tissues in the current study. We suggest that CGA may improve f lap survival because of its antioxidant properties. We also propose to further investigate the EA route for comparing CGA and its metabolites. In summary, CGA enhanced abdominal skin f lap survival based on EA in nondiabetic and diabetic rats. Although there are many limitations in this study, the beneficial effects of CGA in improving f lap survival may be explained by its antioxidant activity. ACKNOWLEDGMENT The authors thank Y. Bagdas for English editing of the manuscript.
REFERENCES 1. Myers B. Understanding flap necrosis. Plast Reconstr Surg. 1986;78:813Y814. 2. Isken T, Ozgentas HE, Gulkesen KH, et al. A random-pattern skin-flap model in streptozotocin diabetic rats. Ann Plast Surg. 2006;57:323Y329. 3. Aydogan H, Gurlek A, Parlakpinar H, et al. Beneficial effects of caffeic acid phenethyl ester (CAPE) on the ischaemia-reperfusion injury in rat skin flaps. J Plast Reconstr Aesthet Surg. 2007;60:563Y568. 4. Bilen BT, Kilinc¸ H, Alaybeyoglu N, et al. Effect of caffeic acid phenethyl ester on survival of axial pattern flaps in rats with ischaemia-reperfusion injuries. Scand J Plast Reconstr Surg Hand Surg. 2006;40:73Y78.
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Chlorogenic Acid in Skin Flap Survival
5. Ozyazgan I, Tuncer A, Yazici C, et al. Reactive oxygen species in experimental ischemic flow-through venous flaps and effects of antioxidants on reactive oxygen species and flap survival. Ann Plast Surg. 2007;58:661Y666. 6. Clifford MN. Chlorogenic acids and other cinnamatesVnature, occurrence and dietary burden. J Sci Food Agric. 1999;79:362Y372. 7. Bagdas D, Cinkilic N, Ozboluk HY, et al. Antihyperalgesic activity of chlorogenic acid in experimental neuropathic pain. J Nat Med. 2013;67: 698Y704. 8. Cinkilic N, Cetintas SK, Zorlu T, et al. Radioprotection by two phenolic compounds: chlorogenic and quinic acid, on x-ray induced DNA damage in human blood lymphocytes in vitro. Food Chem Toxicol. 2013;53:359Y363. 9. dos Santos MD, Almeida MC, Lopes NP, et al. Evaluation of the antiinflammatory, analgesic and antipyretic activities of the natural polyphenol chlorogenic acid. Biol Pharm Bull. 2006;29:2236Y2240. 10. Shimoyama AT, Santin JR, Machado ID, et al. Antiulcerogenic activity of chlorogenic acid in different models of gastric ulcer. Naunyn Schmiedebergs Arch Pharmacol. 2013;386:5Y14. 11. Karthikesan K, Pari L, Menon VP. Combined treatment of tetrahydrocurcumin and chlorogenic acid exerts potential antihyperglycemic effect on streptozotocin-nicotinamide-induced diabetic rats. Gen Physiol Biophys. 2010; 29:23Y30. 12. Chen WC, Liou SS, Tzeng TF, et al. Effect of topical application of chlorogenic acid on excision wound healing in rats. Planta Med. 2013;79:616Y621. 13. Bagdas D, Cam-Etoz B, Inan-Ozturkoglu S, et al. Effects of systemic chlorogenic acid on random-pattern dorsal skin flap survival in diabetic rats. Biol Pharm Bull. 2014;37:361Y70. 14. Gribbe O, Gustafsson LE, Wiklund NP. Transdermally administered nitric oxide by application of acidified nitrite increases blood flow in rat epigastric island skin flaps. Eur J Pharmacol. 2008;578:51Y56. 15. Fromes Y, Liu JM, Kovacevic M, et al. The tetrapeptide acetyl-serine-aspartyllysine-proline improves skin flap survival and accelerates wound healing. Wound Repair Regen. 2006;14:306Y312. 16. Yamashita S, Sugiyama N, Hasegawa K, et al. A novel model for supermicrosurgery training: the superficial inferior epigastric artery flap in rats. J Reconstr Microsurg. 2008;24:537Y543. 17. Cam Etoz B, Isbil Buyukcoskun N, Etoz A, et al. Preventing flap necrosis with adrenomedullin and glucagon like peptide-1. Wounds. 2012;24:29Y35. 18. Komorowska-Timek E, Timek TA, Brevetti LS, et al. The effect of single administration of vascular endothelial growth factor or L-arginine on necrosis and vasculature of the epigastric flap in the rat model. Br J Plast Surg. 2004;57:317Y325. 19. Scalise A, Tucci MG, Lucarini G, et al. Local rh-VEGF administration enhances skin flap survival more than other types of rh-VEGF administration: a clinical, morphological and immunohistochemical study. Exp Dermatol. 2004; 13:682Y690. 20. Lowry OH, Rosebrough NJ, Farr AL, et al. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951;193:265Y275. 21. Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem. 1979;95:351Y358. 22. Ellman GL. Tissue sulfhydryl groups. Arch Biochem Biophys. 1959;82:70Y77. 23. Sun Y, Oberley LW, Li Y. A simple method for clinical assay of superoxide dismutase. Clin Chem. 1988;34:497Y500. 24. Rodriguez de Sotillo DV, Hadley M. Chlorogenic acid modifies plasma and liver concentrations of: cholesterol, triacylglycerol, and minerals in (fa/fa) Zucker rats. J Nutr Biochem. 2002;13:717Y726. 25. McCarty MF. A chlorogenic acid-induced increase in GLP-1 production may mediate the impact of heavy coffee consumption on diabetes risk. Med Hypotheses. 2005;64:848Y853. 26. Carden DL, Granger DN. Pathophysiology of ischaemia-reperfusion injury. J Pathol. 2000;190:255Y266. 27. Khalil AA, Aziz FA, Hall JC. Reperfusion injury. Plast Reconstr Surg. 2006; 117:1024Y1033.
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