Effects of Systemic Chlorogenic Acid on Random-Pattern Dorsal Skin Flap Survival in Diabetic Rats

Abstract

There has been considerable interest in understanding the effects of antioxidants in flap survival during diabetes. Previous studies showed that chlorogenic acid (CGA) exhibits potent antioxidant effects. We aimed to determine the effects of systemic CGA treatment on skin flap survival in an experimental random-pattern dorsal skin flap model in diabetic rats. Twenty-eight male Wistar rats were divided into four groups: phosphate buffered saline (PBS)-treated or CGA-treated nondiabetic rats, PBS-treated or CGA-treated diabetic rats. Diabetes was induced by streptozotocin (45 mg/kg). Caudally based bipedicled dorsal skin flaps were elevated. CGA (100 mg/kg) or PBS (mL/kg; as vehicle) was administered intraperitoneally once daily. On postoperative day 7, flap survival, regional blood perfusion and microangiography were evaluated. The malondialdehyde (MDA), reduced glutathione (GSH), superoxide dismutase (SOD) and nitric oxide (NO) levels were evaluated from the flap tissue. Capillary density and vascular endothelial growth factor (VEGF) expression were assessed. Harmful effects of diabetes on flap survival were observed. CGA attenuated these effects and allowed greater survival and blood perfusion. CGA decreased MDA and NO levels and increased GSH and SOD levels. CGA elevated capillary density and VEGF expression. This study showed that peripherally administered CGA significantly improved flap survival in diabetic and nondiabetic rats.

Skin flaps are widely used in the repair of local tissue loss and the reconstruction of several tissue defects. Flap necrosis is frequently observed in flap tissues in the postoperative period and is an unwanted effect of healing. Many factors are known to play a role in this major complication such as ischemia, inadequate blood flow and disturbed venous drainage.^1–3) Diabetes mellitus is a metabolic disease that causes delayed wound healing and a predisposition to infections. Skin flap surgery under diabetic condition represents a number of difficult pathophysiological problems, and flap necrosis is difficult to treat with currently accepted strategies. Diabetes aggravates flap necrosis and causes/increases oxidative stress in general and tissue-based.^4,5) Potential new therapeutics for improving flap survival and suppressing necrosis and oxidative stress are being investigated.

Ischemic injury participates in the pathophysiology of many surgical conditions, and oxidative and inflammatory reactions occur in the ischemic tissue as a result of the injury response.⁴⁾ Free radicals may play an important role in ischemic damage, and the generation of reactive oxygen radical species (ROS) in skin flap tissue is one of the mechanisms for flap necrosis.^4,6,7) ROS generation is the major cause of lipid peroxidation,⁸⁾ which is aggravated by the presence of diabetes.^4,5) 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 superoxide dismutase (SOD), catalase (CAT) and reduced glutathione (GSH).⁸⁾

Numerous studies have successfully applied antioxidants for protection against flap necrosis and other related complications.^7,9–14) Various natural antioxidants significantly improve flap survival.^11,15–17) Strategic therapeutic approaches with antioxidants have been used to reduce the oxidative stress effects of diabetes.⁵⁾ These findings suggest that antioxidants may modulate the oxidative stress response in flap tissue, especially in diabetic flap surgery.

Chlorogenic acid (5-O-caffeoylquinic acid, CGA) is a phenolic compound that occurs naturally in plants, fruits and vegetables.¹⁸⁾ The antioxidant, anti-inflammatory, analgesic, antiulcerogenic and hypoglycemic effects of CGA have been well described.^19–24) Experimentally, CGA has been reported to exhibit free-radical scavenging and antagonistic effects against lipid peroxidation in different organs.^11,22,25,26) The effects of topical CGA treatment on an excisional wound model have been investigated recently.²⁷⁾

No published studies to date have investigated the healing properties of systemic CGA on flap survival and flap tissue pathophysiology during CGA therapy. CGA may exert better flap survival due to its properties and wound healing effects. We aimed to determine the possible healing effects of systemic CGA treatment on survival in an experimental random-pattern dorsal skin flap model in diabetic and nondiabetic rats.

MATERIALS AND METHODS

Chemicals

CGA was purchased from Acros Organics (Belgium). Streptozotocin, thiobarbituric acid, 5,5′-dithiobis-2-nitrobenzoic acid and 1,1,3,3-tetramethoxypropane were obtained from Sigma Chemical Co. (U.S.A.), and citric acid was obtained from J.T. Baker (U.S.A.). Trichloroacetic acid was acquired from Merck (Germany). Na₂HPO₄·2H₂O and glutathione were obtained from BioShop (Canada). The SOD determination kit was purchased from Sigma Chemical Co. (Switzerland, Catalog Number 19160). The nitric oxide (NO) (NO₂⁻/NO₃⁻) detection kit was purchased from Enzo Life Science Co., U.S.A. (Catalog Number ADI-917-010). Other chemicals were obtained from local commercial sources.

Animals

The study was performed on 28 male Wistar albino rats with ages of 12–16 weeks and weights of 300–350 g at the beginning of the experiments. The rats (supplied by the Experimental Animals Breeding and Research Center, Uludag University) were housed in conventional cages with 1 rat per cage in a temperature- and humidity-controlled room (21±2°C, 50±5%) on a 12-h 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.

Study Plan

The rats were divided into four equally sized groups: phosphate buffered saline (PBS)-treated (as nondiabetic control) or CGA-treated nondiabetic rats and PBS-treated (as diabetic control) or CGA-treated diabetic rats. CGA was administered at a dose of 100 mg/kg, and PBS, which was used as a vehicle to dissolve CGA, was administered to the control groups. The dosage of CGA was selected from the effective dose of our previous study²⁰⁾ and from preliminary experiments for this study (data not shown). The CGA and PBS treatments were started after operation on the surgery day and continued for postoperative six days. Azuma et al. suggested that CGA is not well absorbed from the digestive tract when administered orally to rats, although the compound easily enters blood vessels after intraperitoneal (i.p.) injections.²⁸⁾ We chose i.p. injections instead of oral administration to elicit a stable drug concentration. The treatments were administered i.p. in a 0.5 mL volume. No rats died during the study, and all the rats were sacrificed on postoperative day 7.

Induction of Diabetes and Estimation of Blood Glucose Level

First, all the rats were weighed, and 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, CA, U.S.A.). Experimental diabetes mellitus was induced by a single 45 mg/kg i.p. injection of streptozotocin to overnight fasted rats. Following the induction of diabetes, rats received a solution of 6% sucrose in their drinking water for 24 h. The nondiabetic rats were injected with the vehicle of streptozotocin in an equivalent volume. Four days later, blood glucose assessments were repeated, and the rats with fasting blood glucose levels more than 300 mg/dL were considered to be diabetic.²⁹⁾ The diabetic rats were used six weeks later, and the diabetic status was controlled by assessments of the blood glucose levels six weeks after the streptozotocin administration. During this period, the clinical signs of diabetes mellitus such as polyuria, polyphagia and weight loss were observed in the diabetic rats.

Experimental Dorsal Skin Flap Model

All the surgical procedures were performed under 2.5–3.5% sevoflurane inhaled anesthesia. The surgical area was shaved with an electric razor and manually depilated (Veet® hair removal cream) under light anesthesia one day before the operation. On the surgery day, the surgical area was covered with sterile drapes following iodine antisepsis. A modified McFarlane dorsal skin flap was designed for the experiments.³⁰⁾ A rectangular, caudally based random-pattern flap measuring 4×8 cm was marked on the back of each rat. The distal margin of the flap was located posterior to the scapula. The flap, including the panniculus carnosus, was raised by sharp dissection and sutured back to the original position with 4–0 silk sutures. After surgery, the rats were maintained for 7 d under normal conditions in their own cages.

Evaluation of Flap Survival and Blood Flow

On postoperative day 7, the rats were anesthetized as described above. An image of the last condition of the flap was obtained with a digital camera (Nikon CoolPIX L25; Nikon Corporation, Tokyo, Japan) from a distance of 20 cm. After photography, the flap area was divided into four equal sections and marked as region 1 to 4 from distal to proximal (Fig. 1). A laser Doppler flowmeter (LDF; OxyLab Laser Doppler Flowmeter, Oxford Optronix Ltd., United Kingdom) was used to measure the regional blood perfusion of each area in the flap tissue on postoperative day 7.³¹⁾ The measurements were recorded into the computer as tissue perfusion units (blood perfusion unit, BPU, mL/min/100 g tissue) using the MP30 Data Acquisition System, Biopac Systems Inc., U.S.A.

Fig. 1. Design of Experimental Random-Pattern Dorsal Skin Flap Model

Each flap was 8 by 4 cm. R1–4 indicate the four points for the regional blood perfusion assessment. The yellow shaded rectangular represents the site of the tissue sample for biochemical analyses of oxidative stress markers, and the blue shaded rectangular shows the site of biopsy. (Color images were converted into gray scale.)

After determination of blood flow, the necrotic and viable areas of the total flaps were traced on clear transparent sheets, one rat from each group was separated for microangiography and the others were sacrificed. The sheets were scanned as digital images, and the images were analyzed using Image J software (version 1.44p, National Institutes of Health, Bethesda, MD, U.S.A.) to calculate the percentage of the survival area.³¹⁾ The researchers were blind to the identification of the treatment groups during analysis of the digital images. The total size of each flap tissue and the survival area were measured separately to determine the ratio of flap survival area to the total flap tissue area, and the results were presented as a percentage of the total flap area (area %=[survival area/flap area]×100)

Microangiographic Evaluation

Microangiography was performed as described by Isken et al.³²⁾ A 24-gauge catheter was inserted through the right carotid artery under anesthesia (n=1 in each group). Heparin (250 U/mL) was used to flush the artery. Barium sulfate (25%) in warm water was administered gently with low pressure and was stopped when it began to ooze from the margins of the flaps. After ten minutes, the flaps with the surrounding skin tissue were dissected and X-ray images (Fujifilm FCR Capsula XLII model CR-IR 359, Tokyo, Japan) were obtained by radiography machine (Philips DuoDiagnost Universal R/F, Eindhoven, the Netherlands).

Determination of Oxidative Stress Markers

Flap tissue samples were rapidly collected from the transition area between R1 and R2 (Fig. 1) after the rats were sacrificed (n=6 in each group). The samples were washed well in cold 0.9% NaCl and were immediately frozen at −20°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. The samples were vortexed for 10 s and centrifuged (4000 rpm, 15 min, 4°C). The supernatants were used for analysis. Furthermore, the tissue protein levels were determined in 50 µL of homogenate according to the method of Lowry et al.³³⁾

Malondialdehyde (MDA) and reduced GSH were measured spectrophotometrically (Mannheim Boehringer Photometer 4010). The MDA levels of the flap tissue were determined by the thiobarbituric acid method³⁴⁾ by adding 1 mL of 0.67% thiobarbituric acid solution to 0.5 mL of the supernatant in a glass tube and holding the tube at 100°C for 60 min. After cooling the tubes, the absorbance of the supernatant was read at 546 nm. The results were expressed as pmol/mg protein, and 1,1,3,3-tetramethoxypropane was used as the MDA standard.

The GSH levels were measured according to the Ellman method³⁵⁾ by adding 2 mL of 0.3 M Na₂HPO₄·2H₂O and 0.2 mL of 0.4 mg/mL 5,5′-dithiobis-2-nitrobenzoic acid (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 µmol/mg protein.

Superoxide dismutase (SOD) activity was assessed using a commercial ELISA kit. 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.³⁶⁾ The results were expressed as U/mg protein.

Nitric oxide (NO) production was measured as nitrite (NO₂⁻) accumulation in the flap tissues using an ELISA kit by following the manufacturer’s protocol. Briefly, nitrite was measured using the Griess reaction.³⁷⁾ The results were expressed as pmol/mg protein.

Histopathological Evaluation and Vascular Endothelial Growth Factor (VEGF) Immunostaining

The distal region of the flaps as shown in Fig. 1 was used for the histopathological exam and immunohistochemistry.

Histopathological staining: 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-µM-thick tissue sections, mounted on slides and stained with hematoxylin-eosin (H&E). Minimum of two fields were examined for each slide semiquantitatively under a light microscope at 200× magnification and scored for histopathological changes according to the scale in Table 1. Slides were graded as (0) absent, (1) slight, (2) moderate, (3) marked or (4) extensive for capillary density, congestion, edema, necrosis, infiltration of polymorphonuclear leukocytes (PMNL) and proliferation of fibroblasts, and the average number of findings was noted.

Table 1. Histopathological Evaluation Criteria

Criteria	Capillary density	Congestion	Edema	Necrosis	PMNLs infiltration	Fibroblast proliferation
0 indicates “absent”	No capillary	None detected	No perivascular fluid	Complete healing	0, no PMNL infiltration	No active fibroblast proliferation
1, “slight”	0–5 red blood cell-perfused capillaries	Detectable, rare (< 25%)	Detectable, rare (< 25%)	Complete remodeling of epidermis and dermis	0–5 PMNL infiltration	Thin granulation layer
2, “moderate”	5–10 red blood cell-perfused capillaries	Moderate extent/distribution (25% to <50%)	Moderate extent/distribution (25% to <50%)	Erosive, moderate epidermal and dermal organization	5–10 PMNL infiltration	Granulation layer
3, “marked”	10–20 red blood cell-perfused capillaries	Intensive extent/distribution (50% to <75%)	Intensive extent/distribution (50% to <75%)	Necrotic, but little epidermal and dermal organization	10–20 PMNL infiltration	Thick granulation layer
4, “extensive”	> 20 red blood cell-perfused capillaries	Widespread extent/distribution (75% to 100%)	Widespread extent/distribution (75% to 100%)	Tissue unhealthy and infected	> 20 PMNL infiltration	Very thick granulation layer

Immunohistochemical staining: Tissue sections were stained using commercially available rabbit-anti-human VEGF Ab-1(RB-222-PO) antibodies from NeoMarkers (CA, U.S.A.) and a streptavidin biotin complex (Strept-ABC) method. The tissue sections were dewaxed using xylene and ethanol. Slides in 10 mM citrate buffer were treated in the microwave 4 times of 5 min each at 700 W with the addition of citrate buffer to compensate for evaporation between each 5 min cycle for antigen retrieval. After blocking the endogenous peroxidase activity with 3% H₂O₂ in methanol for 5 min and rinsing in PBS, a protein blocking solution was applied for 10 min. The primary antibody, which was diluted 1 : 100 in PBS (5 µg per slide), was applied for 1 h. The cells were rinsed in PBS, and biotinylated secondary goat-anti-mouse antibody (LabVision, CA, U.S.A.) was applied for 30 min. After incubation with streptavidin-peroxidase (LabVision, CA, U.S.A.) for 30 min, Diaminobenzidine was applied for 15 min for color development. The slides were finally counterstained with Harris hematoxylin and mounted with Entellan®. Hemangiosarcoma was used as a positive control. Negative controls were analyzed by replacing the primary antibody with PBS. The number of endothelial cells expressing VEGF was counted in two fields of 400× magnification.

Statistical Analysis

Data are expressed as the mean±S.E.M. (standard error of the mean) in all cases. Each group contained 6–7 rats. Data were analyzed using one-way ANOVA followed by the Student–Newman–Keuls test. A p value of less than 0.05 was considered significant.

RESULTS

Effect of Chlorogenic Acid (CGA) on Blood Glucose Level and Body Weight Changes

As shown in Table 2, the blood glucose levels were higher in the diabetic rats than the nondiabetic rats after streptozotocin treatment. Postoperative CGA treatments did not affect the blood glucose level in diabetic rats. Table 2 shows that the nondiabetic rats gained weight continuously but not significantly during the course of the study. The diabetic groups had significantly lower weight gain during the study. CGA treatment did not change the body weight in all the rats.

Table 2. Average Blood Glucose Levels and Body Weights in Nondiabetic and Diabetic Groups

Groups	n	Day 0		Day 4		Day 42		Postoperative day 7
		BGc)	BWd)	BGc)	BWd)	BGc)	BWd)	BGc)	BWd)
Nondiabetic
PBSa)	7	84.4±2.3	327.3±15.5	88.8±3.2	331.7±13.8	85.5±2.8	357.7±7.5	84.4±1.5	351.3±8.2
CGAb)	7	88.3±5.3	328.0± 14.9	86.0±3.4	330.2±13.7	82.6±1.9	354.9±9.8	84.0±2.4	345.0±11.7
Diabetic
PBSa)	7	87.3±3.7	320.1±12.8	406.7±16.8***	302.9±11.2	368.9±22.9***	288.1±4.7*	386.3±16.8***	277.4±3.1**
CGAb)	7	88.9±3.3	334.1±6.8	410.1±17.3***	318.7±14.0	409.7±13.4***	299.9±8.0**	393.6±15.8***	294.4±8.1**

Data are presented as means±S.E.M. a) PBS indicates “phosphate buffered saline as vehicle control”; b) CGA, “chlorogenic acid”; c) BG, “average of blood glucose level”; d) BW, “average of body weight.” * p<0.05 vs. Day 0; ** p<0.01 vs. Day 0; *** p<0.001 vs. Day 0.

Effect of CGA on Flap Survival and Blood Flow

A macroscopically photographic presentation of the flaps on the seventh postoperative day is shown in Fig. 2. Necrotic areas of the flap were dark in color with a rough texture, while the survival flap area was a normal color and normal texture. The flap survival ratio is shown in Fig. 3. Flap necrosis in the PBS-treated diabetic rats was significantly more pronounced compared with the PBS-treated nondiabetic rats. The necrotic areas of the flaps were smaller and flap survival rate was significantly increased in the CGA treatment groups than the PBS-treated groups. Additionally, diabetes caused significantly deficient blood flow in the flap tissue on postoperative day 7. The groups treated with CGA showed a statistically significant increase in blood flow in regions 1 and 2 (Figs. 4 A, B). In the diabetic rats, CGA also increased blood flow in region 3. We also measured regional blood flow after the first CGA treatment on the day of surgery, but we did not observe any difference among groups (data not shown).

Fig. 2. Photographic Presentations of Flaps

A, phosphate buffered saline (PBS)-treated nondiabetic; B, chlorogenic acid (CGA)-treated nondiabetic; C, PBS-treated diabetic and D, CGA-treated diabetic rats.

Fig. 3. The Mean Flap Survival Rate (%)

Abbreviations are indicated in the footnote of Fig. 2. Data are expressed as the mean±S.E.M. of 7 animals for each group. Statistical analysis was performed using one-way ANOVA followed by Student–Newman–Keuls test. * p<0.05 and *** p<0.001 are significantly different from its vehicle group. ## p<0.01 is significantly different from the nondiabetic PBS-treated group.

Fig. 4. Regional Blood Perfusion in the Skin Flaps

A, nondiabetic rats; B, diabetic rats. Abbreviations are indicated in the footnote of Fig. 2. Data are expressed as the mean±S.E.M. of 7 animals for each group. Statistical analysis was performed using one-way ANOVA followed by Student–Newman–Keuls test. * p<0.05, ** p<0.01 and *** p<0.001 are significantly different from its vehicle group. ## p<0.01 is significantly different from the nondiabetic PBS-treated group.

Microangiographical Results

We observed no noticeable difference in vessel quantity in microangiography between nondiabetic and diabetic groups. A slight increase was found in the quantity of vessels in nondiabetic and diabetic CGA-treated rats, although we did not perform a statistical analysis (Fig. 5).

Fig. 5. Photographs of Microangiographies

A, PBS-treated nondiabetic; B, CGA-treated nondiabetic; C, PBS-treated diabetic and D, CGA-treated diabetic rats. Abbreviations are indicated in the footnote of Fig. 2.

Effect of CGA on Oxidative Stress Markers

The MDA and NO levels were significantly higher in the PBS-treated diabetic rats compared with PBS-treated nondiabetic rats, while the GSH levels were reduced. CGA significantly decreased the MDA and NO levels in both nondiabetic and diabetic rats. Additionally, CGA significantly increased the GSH levels in diabetic rats. The SOD levels did not show significant difference between the PBS-treated nondiabetic and diabetic rats. However, CGA significantly increased the SOD activity in both nondiabetic and diabetic groups (Table 3).

Table 3. The Antioxidant Status and Nitric Oxide Levels of Flap Tissues in Nondiabetic and Diabetic Groups

Groups	n	MDAc) (pmol/mg protein)	GSHd) (µmol/mg protein)	SODe) (U/mg protein)	NOf) (pmol/mg protein)
Nondiabetic
PBSa)	6	240.0±38.6	6.1±0.2	208.5±25.9	245.0±60.4
CGAb)	6	129.1±28.7#	8.3±1.5	326.3±48.2#	78.5±4.2##
Diabetic
PBSa)	6	874.8±190.7**	3.2±0.7**	234.4±13.2	450.0±26.9*
CGAb)	6	119.0±55.7##	13.9±4.3#	367.7±43.0#	101.0±17.7###

Data are presented as means±S.E.M. a) PBS indicates “phosphate buffered saline as vehicle control”; b) CGA, “chlorogenic acid”; c) MDA, “malondialdehyde”; d) GSH, “reduced glutathione”; e) SOD, “superoxide dismutase”; f ) NO, “nitric oxide.” * p<0.05 vs. nondiabetic PBS; ** p<0.01 vs. nondiabetic PBS; # p<0.05 vs. its control; ## p<0.01 vs. its control; ### p<0.001 vs. its control

Effect of CGA on Histopathological Changes and VEGF Immunostaining

H&E Results: CGA treatment caused neovascularization by increasing capillary density in both nondiabetic and diabetic rats (Table 4, Fig. 6). The capillary density was higher in the nondiabetic CGA group compared with the diabetic CGA group. Similar levels of congestion, edema and PMNLs infiltration were observed in each group. Congestion and PMNLs infiltration were higher in the nondiabetic groups. Fibroblastic proliferation was aligned unlike congestion and infiltration row. The CGA-treated diabetic group showed the highest fibroblast proliferation. The administration of CGA in nondiabetic and diabetic rats decreased the necrosis compared with PBS-treated rats.

Table 4. Semiquantitative Histopathological Findings of Flap Tissues in Nondiabetic and Diabetic Groups

Groups	n	Capillary density	Congestion	Edema	Necrosis	PMNLsc)infiltration	Fibroblastproliferation
Nondiabetic
PBSa)	6	2.6	2.6	2.6	2.0	2.8	1.6
CGAb)	6	3.2	2.4	2.2	0.2	2.6	2.8
Diabetic
PBSa)	6	1.2	2.2	2.6	2.6	2.4	3.2
CGAb)	6	2.8	2.0	2.8	0.4	2.2	4.0

Histopathological evaluation criteria are indicated in the Table 1. Data are presented as means obtained from each group. a) PBS indicates “phosphate buffered saline as vehicle control”; b) CGA, “chlorogenic acid”; c) PMNLs, “polymorphnuclear leukocytes.”

Fig. 6. Histopathological Appearance of Flap Tissues

A, PBS-treated nondiabetic; B, CGA-treated nondiabetic; C, PBS-treated diabetic and D, CGA-treated diabetic rats. Abbreviations are indicated in the footnote of Fig. 2. Arrows indicate formation of new capillaries (by hematoxylin and eosin, 200× magnification).

Immunohistochemical results: The average number of positive stained endothelial cells with VEGF increased significantly in the CGA-treated groups compared with the PBS-treated groups (Figs. 7, 8). Diabetes slightly reduced, but the CGA treatment increased the positive staining of endothelial cells.

Fig. 7. Immunohistochemical Staining for Vascular Endothelial Growth Factor (VEGF) in Sections of Flap Tissues

A, PBS-treated nondiabetic; B, CGA-treated nondiabetic; C, PBS-treated diabetic and D, CGA-treated diabetic rats. Abbreviations are indicated in the footnote of Fig. 2. Arrows indicate positively stained endothelial cells for VEGF (400× magnification).

Fig. 8. Immunohistochemical Analysis of Vascular Endothelial Growth Factor (VEGF) Expression in Skin Flaps

Abbreviations are indicated in the footnote of Fig. 2. Data are expressed as the mean±S.E.M. of 6 animals for each group. Statistical analysis was performed using one-way ANOVA followed by Student–Newman–Keuls test. * p<0.05 is significantly different from the vehicle group.

DISCUSSION

The present study shows for the first time that systemic administration of CGA promotes significant healing in an experimental random-pattern dorsal skin flap model in nondiabetic and diabetic rats. The CGA-treated nondiabetic and diabetic groups demonstrated significantly greater flap survival, blood perfusion and microangiographical results compared with the PBS-treated groups (Figs. 2, 3, 4, 5). CGA significantly decreased the MDA and NO levels and increased the GSH and SOD levels in tissue samples of diabetic rats (Table 3). Histopathological examinations revealed increased capillary density and VEGF expression (Fig. 8, Table 4). CGA did not exhibit antidiabetic effects by day 7 of treatment or alter the body weight (Table 2), but CGA exhibited more potent healing activity in diabetic rats compared with nondiabetic rats.

The adverse effects of diabetes on flap survival are well known. The most common complication of diabetes in surgical flaps is increased partial-flap necrosis.^32,38) In this study, the flap survival of the PBS-treated diabetic group was significantly lower than the PBS-treated nondiabetic group. Our results are consistent with other studies.

Recently, the therapeutic potentials of various natural antioxidants on survival in flap tissues are increasingly studied.^16,39) CGA, a natural phenolic compound found in human diet, has been reported to exhibit potent antioxidant and anti-inflammatory activities.^18–21,40) Hypoglycemic effects of CGA has been shown in diabetic rats.^23,41) CGA 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.²⁷⁾ In accordance with its wound healing potential, CGA enhanced flap survival in our study. Caffeic acid phenethyl ester, which is an ester form of CGA metabolites, was shown to exhibit beneficial effects on the survival of dorsal skin flaps and protective effects against I/R injury by surgical skin flap.^16,42) Additionally, caffeic acid phenethyl ester partly accelerates cutaneous full-thickness wound healing by its antioxidant and ROS-scavenging capabilities.⁴³⁾ These findings suggest that the effect of CGA in our study may be a result of CGA action itself and/or the action of its metabolites, such as caffeic acid. Further studies are needed to clarify this hypothesis.

Natural phenolics have been reported to exhibit free radical scavenging and antagonistic effects against lipid peroxidation in in vitro and in vivo models.^11,23,40,41) Yun et al. showed that CGA exhibits protective effects against I/R injury in rat liver by its antioxidant and anti-inflammatory properties.⁴⁰⁾ CGA has been shown to suppress oxidative stress, as indicated by hepatic lipid peroxidation and GSH levels.⁴⁰⁾ Additionally, CGA has been demonstrated to decrease the MDA levels in skin microsomes in vitro.¹¹⁾ Caffeic acid phenethyl ester was shown to exhibit healing effects on flap survival by decreasing the MDA and NO levels and increasing the GSH and SOD levels in flap tissue.^16,42) In our study, the strong radical scavenger CGA significantly suppressed the MDA levels, increased the GSH and SOD levels of the flap tissues. We suggest that CGA quantitatively and effectively improved flap survival due to its antioxidant properties.

NO, an inorganic free radical, is involved in the biochemical processes of the flap tissue. NO is important in the regulation of vascular tone and blood flow and in the inhibition of platelet and neutrophil aggregation.⁴⁴⁾ The generation of superoxide anion (O₂⁻) and NO increases in diabetes mellitus and is closely related to the development of diabetic complications. NO can be destructive and cytotoxic or protective depending on the oxidation-reduction state of the molecule.^44,45) Externally NO has been demonstrated to exhibit beneficial effects in wound repair. Although exogenous NO therapy improves wound healing, the exact mechanisms of NO activity on healing parameters are still unknown.⁴⁶⁾ Previous studies have revealed that an adequate amount of NO is necessary, and overproduction will cause negative effects.^46–48) Low NO concentrations stimulate cell division, while high concentrations are cytostatic.^49,50) In addition, excessive endogenous inducible nitric oxide synthase production delays wound healing. Particularly, NO is expressed ubiquitously in inflammation, and as a result of its increased production; high levels of NO are produced in response to inflammatory stimuli and mediate pro-inflammatory and harmful effects.^46,47,51) In flap surgery, exogenous NO therapy enhances flap survival and maintains circulation in the skin flap periphery.^52,53) On the other hand increased endogenous NO levels may cause inadequate blood perfusion, which is the major cause of flap necrosis, and diabetes may aggravate the situation.^16,44) In the current study, the NO levels were higher in the flap tissues of PBS-treated rats, and diabetes significantly increased the level of NO. However, CGA significantly decreased the NO levels in all the flap tissues. Moreover, the regional blood perfusion assessments showed that CGA induces greater blood flow. In the light of these observations and the dual action of NO, one of the mechanisms of CGA in improving blood perfusion and flap survival may be its inhibitory effect on endogenous NO production in flap tissue. Caffeic acid, one of the major metabolite of CGA, also has been shown to have beneficial effects on tissue NO levels in skin flaps. In correlation with our results, caffeic acid decreased the NO levels of skip flap tissue.¹⁶⁾

Chronic hyperglycemia induces pathological changes in tissues, delays wound healing and increases the predisposition to infections.^5,54,55) CGA has been reported to modulate blood glucose levels and exhibits protective effects against tissue changes in diabetes.⁴¹⁾ CGA has been shown to exhibit hypoglycemic activity at a 27 mg/kg dosage in streptozotocin diabetic rats within 11 d²⁴⁾ or 5 mg/kg/day orally for 45 d in streptozotocin-nicotinamide-induced diabetes.²³⁾ CGA 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.²³⁾ Interestingly, we did not observe a significant difference in the blood glucose level in diabetic rats in this study. CGA (100 mg/kg) exhibited beneficial effects on the complications of hyperglycemia but did not induce hypoglycemia after 7 d of administration. The different therapeutic timetable used in the other experiments may explain the contradicting results.

The decay of a tissue or the delay of the healing in diabetes mellitus is characterized by impaired cellular infiltration and granulation tissue formation, elevated ROS levels, diminished blood perfusion, reduced collagen organization, changes in blood viscosity and reduced angiogenesis.^5,54,55) In the current study, the harmful effects of diabetes were also detected in our histopathological, immunohistochemical, Doppler and microangiographical assays. The semiquantitative histopathological findings of our study showed enhanced capillary density and fibroblast proliferation and decreased necrosis in peripherally CGA-treated rats compared with the PBS-treated rats. Congestion, edema and PMNLs infiltration exhibited similar levels in each group. It is well known that VEGF induces the migration and proliferation of endothelial cells and enhances vascular permeability. VEGF shows an ability of increasing of angiogenesis on wound healing. Therefore, physiological VEGF plays a crucial role in tissue repair.⁵⁶⁾ Increased VEGF expression showed improved microvascular perfusion and enhanced neovascularization in skin flap surgeries.^39,56,57) It has been reported that therapeutic potentials of natural antioxidant epigallocatechin gallate on flap survival by inducing VEGF expression.³⁹⁾ On the other hand, same compound inhibits pathological VEGF in cancerous tissue.⁵⁸⁾ Correspondingly, a recently published study found that CGA demonstrates a dose dependent protective effect on diabetic retinopathy in experimentally induced diabetic rats by decreasing pathological VEGF and occludin levels in retina.⁵⁹⁾ Diabetic retinopathy is one of the complications of diabetes that results from excess blood vessel growth into the vitreous fluid of the eye and pathological VEGF is critically involved in retinal angiogenesis.⁶⁰⁾ The findings in the present study showed that CGA treatment led to an increase in VEGF expression. Based on the findings of both studies, CGA shows different activities in physiological and pathological VEGF expressions as reported for epigallocatechin gallate. CGA has strong anti-inflammatory and antioxidant activities.^18–21) While the exact mechanisms for VEGF expression process are unclear, these activities may involve in it for flap tissue healing. The mechanisms in the effect of CGA on VEGF expression in different situations need further examinations.

Our enhanced VEGF expression results are also consistent with the assessments of regional blood perfusion. CGA treatment resulted in significantly greater perfusion, especially in the diabetic rats. A slight increase was found in the quantity of vessels in microangiography in nondiabetic and diabetic CGA-treated rats, although we did not perform a statistical analysis. In the light of these observations, we suggest that the neovascularization effects of CGA may contribute to the mechanisms of the healing effect on flap survival.

In conclusion, this study showed for the first time that systemic administration of CGA enhanced skin flap viability in an experimental random-pattern dorsal skin flap model in rats. The beneficial effects of CGA in improving flap survival may be explained by the following mechanisms: neovascularization, increased blood perfusion, antioxidant effects on oxidative parameters, free radical scavenger effects and protective lipid peroxidation effects. Upcoming studies may clarify the healing effects of CGA supplementation or dietary CGA in vegetable foods on skin flaps.

Acknowledgment

The authors thank Research Foundation of Uludag University for financial support (Grant no. KUAP (T) 2012/37). The manuscript was edited by American Journal Experts (AJE).

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