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Introduction

Surgical skin flaps are often used in plastic and reconstructive surgery to repair defects resulting from trauma, congenital defects, cancer excision or additional mechanisms. Partial or complete skin flap necrosis is a common problem encountered postoperatively. Inadequate blood perfusion is thought to be the main factor resulting in flap necrosis. Several methods have been used in an attempt to increase blood supply and tissue perfusion in compromised tissues, including therapeutic angiogenesis using exogenous growth factors such as vascular endothelial growth factor (VEGF) and fibroblast growth factor (1,2), as well as ischemic preconditioning (3) and surgical delay (4). However, these methods are not widely used in the clinic due to lack of validation of efficacy and side effects.

The density of vascular structures affects blood circulation and perfusion to the skin flap. The skin flap transplantation process is associated with tissue injury, and the regeneration of blood vessels (neovascularization) is required to revascularize the injured tissue. Stromal cell derived factor-1 (SDF-1) is a constitutively expressed and inducible chemokine that regulates multiple physiological processes including organ repair and tumor development. The biological effects of SDF-1 are mediated by the specific receptor CXCR4, which belongs to a family of G-protein-coupled receptors, and is widely and constitutively expressed by numerous hematopoietic and endothelial cells (5). It has been demonstrated that SDF-1 and CXCR4 are constitutively expressed in a wide range of tissues, including brain, lung, liver, skin and bone marrow (6). In addition, recent studies have shown that the expression of SDF-1 in a large number of tumors and injured tissues strongly suggests that the activation of CXCR4 is involved in promoting neovascularization (7,8).

Numerous studies have demonstrated the efficacy of hyperbaric oxygen (HBO) treatment on enhancing skin flap and skin graft survival by increasing arterial oxygen tension, reducing ischemia-reperfusion injury, enhancing host response to local infections, and stimulating neovascularization and tissue growth (4,9–11). The present study aimed to assess whether HBO treatment is able to improve skin flap survival and neovascularization, whether the expression of SDF-1 and CXCR4 in skin flaps may be induced by HBO treatment and whether neovascularization is affected by the expression of SDF-1 and CXCR4.

Materials and methods

Experimental animals

All the experiments were performed in accordance with the ethical guidelines determined by the Committee for the Purpose of Control and Supervision of Experiments on Animals at the Capital Medical University (Beijing, China). Forty healthy adult male Sprague Dawley rats (weighing, 250–300 g at the beginning of the study) were used. Rats were maintained at 25±1.0°C with a 12/12-h light/dark cycle and received food and water ad libitum.

Experimental groups

Forty rats were randomly assigned to one of the following groups (n=8 per group): i) sham-operated (SH group); ii) ischemia followed by reperfusion and analysis on the third and fifth day postoperatively (IR3d and IR5d groups, respectively); and iii) ischemia followed by reperfusion and HBO treatment and analysis on the third and fifth day postoperatively (HBO3d and HBO5d groups, respectively).

Epigastric pedicle skin flap model

The epigastric pedicle skin flap model used in this study has been described previously (12), with a modification in flap design. Procedures were performed aseptically under anesthesia using intraperitoneal injections of 350 mg/kg 10% chloral hydrate. Rats were fixed on the operating table, the abdomen was shaved and washed before the single inferior epigastric vessel pedicle skin flaps (9×6 cm) were obtained. Skeletonization of the right inferior epigastric artery and vein pedicle was performed, while the contralateral inferior epigastric vessel was ligated with sutures and the feeding vessel was clamped using a microvascular clamp to achieve ischemia. For reperfusion, the microvascular clamp was removed 3 h later and blood flow was restored. The skin flaps were repositioned above a silicone sheet (the same area as the flap) with continuous 5-0 monofilament nylon sutures to prevent vascular supply other than that of the pedicle. The sham-operated group underwent the same surgery but the rats were not exposed to ischemia. All the rats received a single dose of 0.8 mg/g intramuscular penicillin sodium postoperatively.

HBO treatment

In the HBO3d and HBO5d groups, the rats were placed into a custom-made pressure chamber of transparent acrylic plastic (701 Space Research Institute, Beijing, China) immediately following surgery and received 1 h of HBO treatment in a 2.0 ATA chamber in 100% O2 twice per day (8-h intervals) for 3 days and then daily for 2 consecutive days. Compressed air was administered at a rate of 1 kg/cm2/min into the 2.0 ATA chamber including 100% O2 and maintained for 60 min. The chamber was flushed with 100% O2 at a rate of 5 l/min to avoid CO2 accumulation and decompression was performed at 0.2 kg/cm2/min. During HBO exposure, O2 and CO2 content were continuously monitored and maintained at ≥98% and ≤0.03%, respectively, while the chamber temperature was maintained between 22 and 25°C. To minimize the effects of diurnal variation, HBO exposure was initiated at approximately 8 am and 4 pm. Additionally, in the SH, IR3d and IR5d groups, rats were treated with normobaric air in a 1.0 ATA chamber with 21% O2 at an ambient temperature of 22–25°C, postoperatively.

Flap measurements

Evaluation of the skin flaps occurred on the third and fifth day postoperatively. The survival area of the skin flaps was determined based on appearance, color and texture. Viability of the flaps was calculated using Image-Pro Plus software (version 6.0, Media Cybernetics LP, Silver Spring, MD, USA). The results were expressed as percentages relative to the total flap surface area.

Histological analysis

The skin flaps were evaluated on the third and fifth day following surgery. For each skin flap, 3–4 mm-sectioned tissue blocks from viable regions were fixed in 10% formalin and embedded in paraffin for hematoxylin and eosin staining. Microvessel density (MVD) was measured by two double-blinded pathologists using the ‘hot spot’ method. Briefly, five areas with the highest accumulation of small vessels were identified under a low magnification (x100) in each tissue section. These areas were then examined under a high magnification (x400) and microvessels were counted in five high-power fields. Blood vessels with muscular layers were excluded.

Immunohistochemical staining

Monoclonal antibody analysis was performed on the third and fifth day following surgery. For each skin flap, sectioned tissue blocks (3–4 mm) obtained from the viable portion of the flap were preserved in 10% formalin. Tissue sections (4 μm) were obtained and embedded in paraffin for immunohistological analysis. The monoclonal antibodies anti-SDF-1 (1:100) and anti-CXCR4 (1:500) (both from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), were used for immunohistochemical staining. The proportion of positive cells in at least 10 areas in each section were analyzed under a high magnification (x400) by two double-blinded pathologists.

Protein preparation

Skin flap tissues were frozen in liquid nitrogen and then stored at −80°C until analysis. The tissues were homogenized in ice-cold isolation solution containing 250 mmol/l sucrose, 10 mmol/l triethanolamine, 1 μg/ml leupeptin and 0.1 mg/ml phenylmethylsulfonyl fluoride. Homogenates were centrifuged at 15,000 × g for 10 min at 4°C to separate the incomplete homogenized tissue. The supernatants were obtained and the protein concentrations were measured using a protein assay kit (Beijing Sunbio Biotech Co., Ltd., Haidian, China). For deglycosylation of proteins, an N-glycosidase F Deglycosylation Kit (Roche Diagnostics GmbH, Mannheim, Germany) was used.

Western blot analysis

Total proteins (50 μg/sample) were diluted in 5X loading buffer (0.25 mol/l Tris HCl, pH 6.8; 10% sodium dodecyl sulfate; 0.5% bromophenol blue; 50% glycerol; and 0.5 mol/l dithiothreitol) and then heated to boiling point for 5 min. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was carried out on 12% gradient gels. The proteins were electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes that had been previously treated with methanol and blocked for 1 h at room temperature in TBST (Tris-buffered saline containing 0.1% Tween-20) containing 5% non-fat dry milk. PVDF membranes were then incubated overnight at 4°C with anti-SDF-1 (1:100) and anti-CXCR4 antibodies (1:500) (Santa Cruz Biotechnology, Inc.) in TBST containing 5% nonfat dry milk. Membranes were washed in TBST and incubated with horseradish peroxidase-labeled anti-rabbit antibody (1:3,000, Santa Cruz Biotechnology, Inc.) for 2–3 h at room temperature. Blots were developed with enhanced chemiluminescence agents (ECL Plus, Beijing Sunbio Biotech Co. Ltd.) prior to being exposed to X-ray. To confirm equivalent loading of samples, the same membranes were incubated with anti-β-actin antibody (1:300, Santa Cruz Biotechnology, Inc.) and visualized using ECL, as mentioned earlier. For quantification, western blotting films were scanned using a Minolta scanner (Konica Minolta, Inc.) and Adobe Photoshop software. The labeling density was quantitated using LabWorks software (UVP Inc., Upland, CA, USA). The value of the relative density of SDF-1 and CXCR4 bands was normalized to the density of actin to represent the expression of SDF-1 and CXCR4 proteins.

Statistical analysis

Statistical analysis was performed using SPSS software, version 15.0 (SPSS, Chicago, IL, USA). Quantitative data were expressed as the mean ± standard deviation. One-way analysis of variance procedures were used to test the differences in MVD, SDF-1, CXCR4 and the skin flap survival area. The t-test was used to determine the differences in skin flap survival between the HBO3d and HBO5d groups. Relationships between MVD and expression of SDF-1 and CXCR4 were analyzed by calculating the Pearson product-moment correlation coefficient. P<0.05 was considered to indicate a statistically significant difference.

Results

Flap measurements

The regions of survival and necrosis of skin flaps in different groups were clearly demarcated. The surviving skin appeared pink to white, was tender, normal in texture and bled when cut with a scalpel. By contrast, the necrotic skin was black, rigid, abnormal in texture and did not bleed when cut. Rats in the SH group showed an average viable area of 77.5%, compared with that of the IR3d (43.0%) and IR5d (32.2%) groups. The average viable area was significantly increased in the HBO3d (54.7%) and HBO5d (64.7%) groups compared with that of the IR groups (P<0.01). These results indicate that HBO treatment significantly improved skin flap survival. Additionally, when the number of HBO treatments increased from 3 to 5 days, the survival area of skin flaps also significantly increased (P<0.05) (Fig. 1).

Figure 1 Viable area of the skin flaps was compared in each group. Flap survival in the IR and HBO groups was significantly lower than that in the SH group (**P<0.01). Regardless of the number of HBO treatments, flap survival was significantly lower in the IR groups compared with the HBO groups (##P<0.01). Significant flap survival was demonstrated in the HBO5d group when compared with HBO3d group (▲P<0.05). IR, ischemia reperfusion; HBO, hyperbaric oxygen; SH, sham-operated.

Flap neovascularization measurements

To evaluate whether neovascularization varied among the different groups, we measured MVD in the skin flaps and found that MVD was significantly increased in the groups treated with HBO (HBO3d, 8.5±2.9 and HBO5d, 10.0±2.9), compared with that of the IR groups (IR3d, 5.5±1.8 and IR5d, 3.8±1.3) (Fig. 2). These results demonstrate that HBO treatment increased neovascularization in the skin flaps of rats.

Figure 2 Vascularization in the skin flaps of rats. MVD was defined as the total number of microvessels divided by the number of microvessels observed under a high-power field microscope (magnification, ×400) in the (A) IR5d, (B) HBO5d and (C) SH groups. (D) MVD in the IR and HBO groups was significantly lower than that in the SH group (**P<0.01 and *P<0.05). Compared with the same day IR groups, MVD was significantly increased in the HBO groups (##P<0.01 and #P<0.05). MVD, microvessel density; IR, ischemia reperfusion; HBO, hyperbaric oxygen; SH, sham-operated.

Immunohistochemical staining of SDF-1 and CXCR4

Immunohistochemical staining showed that cells were positive for brown particles in the cell membrane, cytoplasm and nucleus. The expression of SDF-1 and CXCR4 in the IR groups was higher than that in the SH group (P<0.05 and P<0.01, respectively). Moreover, the expression of SDF-1 and CXCR4 in the HBO treatment groups was significantly higher than that in the IR groups (P<0.01) and the SH group (P<0.01) (Figs. 3 and 4). These results suggest that HBO treatment significantly increased the expression of SDF-1 and CXCR4 in the skin flaps of rats.

Figure 3 Immunohistochemical staining of SDF-1 in each group. The proportion of positive cells was analyzed under a high magnification (x400) in the (A) IR5d, (B) HBO5d and (C) SH groups. (D) SDF-1-positive cells in the IR and HBO groups were significantly higher than those in the SH group (*P<0.05 and **P<0.01). Compared with the same day IR groups, SDF-1-positive cells were significantly increased in the HBO groups (##P<0.01). IR, ischemia reperfusion; HBO, hyperbaric oxygen; SH, sham-operated.

Figure 4 Immunohistochemical staining of CXCR4 in each group. The proportion of positive cells were analyzed under a high magnification (x400) in the (A) IR5d, (B) HBO5d and (C) SH groups. (D) CXCR4-positive cells in the IR and HBO groups were significantly higher than those in the SH group (**P<0.01). Compared with the same day IR groups, CXCR4-positive cells were significantly increased in the HBO groups (##P<0.01). IR, ischemia reperfusion; HBO, hyperbaric oxygen; SH, sham-operated.

Western blot analysis of SDF-1 and CXCR4

Western blot analysis identified that the expression of SDF-1 proteins in the HBO3d (0.69±0.17) and HBO5d groups (0.81±0.10) was significantly increased compared with that in the IR3d (0.47±0.12) and IR5d groups (0.41±0.11) (P<0.05 and P<0.01, respectively). The expression of CXCR4 proteins in the HBO3d (0.67±0.16) and HBO5d groups (0.84±0.17) was also significantly increased compared with that in the IR3d (0.41±0.13) and IR5d groups (0.38±0.09) (P<0.01). The expression of SDF-1 and CXCR4 in the HBO groups was significantly higher than that in the SH group (SDF-1, 0.33±0.11; CXCR4, 0.31±0.08) (P<0.01) (Fig. 5). These results support the previous findings that HBO treatment significantly increased the expression of SDF-1 and CXCR4 proteins in the skin flaps of rats.

Figure 5 (A) Immunoblot analysis of SDF-1 and CXCR4 in each group. Western blot analysis of (B) SDF-1 and (C) CXCR4 in the skin flap tissue of each group. Data are presented as the mean ± standard deviation. **P<0.01, HBO groups versus SH group; compared with the same day IR groups, SDF-1 and CXCR4 were significantly increased in the HBO groups (##P<0.01 and #P<0.05). HBO, hyperbaric oxygen; SH, sham-operated; IR, ischemia reperfusion.

Positive correlation between neovascularization and SDF-1 and CXCR4 expression

Pearson’s correlation analysis demonstrated that at the protein level, there was a positive correlation between neovascularization (MVD) and the expression of SDF-1 and CXCR4 in the skin flaps of rats treated with HBO (Table I).

Table I Pearson’s correlation analysis between neovascularization (MVD) and SDF-1 and CXCR4 expression in the skin flap of rats (n=6 per group) treated with HBO.

Table I

Pearson’s correlation analysis between neovascularization (MVD) and SDF-1 and CXCR4 expression in the skin flap of rats (n=6 per group) treated with HBO.

Factors	Immunohistochemical staining				Immunoblot analysis
	3 days		5 days		3 days		5 days
MVD	SDF-1	CXCR4	SDF-1	CXCR4	SDF-1	CXCR4	SDF-1	CXCR4
R	0.851a	0.838a	0.914a	0.835a	0.879a	0.821a	0.829a	0.866a
P	0.032	0.037	0.011	0.039	0.021	0.045	0.041	0.026

a P<0.05, compared with P.

{ label (or @symbol) needed for fn[@id='tfn2-mmr-08-04-1118'] } MVD, microvessel density

Discussion

Improving the survival rate of skin flaps is one of the main aims in plastic surgery. A number of studies have demonstrated the beneficial effects of HBO treatment on improving survival of compromised skin grafts and flaps, including the venous flap, the tubed pedicle flap, the composite graft and the random pattern skin flap (10,11,13–15). However, the detailed mechanisms involved are not fully understood. In the present study, we aimed to characterize the effects of HBO treatment on neovascularization and skin flap survival, as well as investigate its mechanism at a molecular level by analyzing the expression of angiogenic mediators, such as SDF-1 and CXCR4 in the skin flaps of rats.

Observations by Ju et al(16) showed that HBO treatment may increase angiogenesis of the expanded skin and subsequently increase skin flap survival. HBO treatment increases neovascularization through angiogenic stimulation, resulting in blood vessel formation from local endothelial cells, and by stimulating systemic stem/progenitor cells to differentiate into blood vessels (17). At present, MVD assessment is one of the most reliable methods of measuring angiogenic activity (18). Consistent with previous studies, the present study measured MVD in skin flap biopsies and findings indicated that MVD was significantly increased in the HBO treatment groups compared with that of the IR groups. These findings demonstrate that HBO treatment increased neovascularization in the skin flaps. To the best of our knowledge, this study analyzed for the first time the concentration of SDF-1 and CXCR4 in the skin flaps of rats, as well as the effects of HBO treatment on the expression of SDF-1 and CXCR4. We found that postoperative administration of HBO resulted in a high expression of SDF-1 and CXCR4 in skin flaps. In addition, Pearson’s correlation analysis indicated a positive correlation between neovascularization and the expression of SDF-1 and CXCR4 in the skin flaps of rats treated with HBO. The results of this study provide a theoretical basis for broadening the clinical application of HBO treatment.

In the present study, the expression of SDF-1 and CXCR4 in the IR groups was higher than that in the SH group, and compared with that of the IR groups, the expression of SDF-1 and CXCR4 was much higher in the HBO groups. Hypoxia initiates neovascularization by inducing angiogenic factor expression, but cannot sustain it. A threshold level of oxygenation is required to support the metabolic needs of tissue remodeling. Acute hypoxia facilitates the angiogenic process (19), while chronic hypoxia impairs wound angiogenesis (20). Sustained hypoxia results in the dysfunction of tissues and cell death. SDF-1 is upregulated in hypoxic or ischemic tissues, such as arteries (21) and brain (22). Moreover, hyperoxia induces the expression of SDF-1 and CXCR4 via the upregulation of VEGF expression (23,24). In addition, Salvucci et al(25) demonstrated that endothelial expression of SDF-1 and CXCR4 may be reduced by inflammatory cytokines, such as tumor necrosis factor-α and interferon-γ. As HBO treatment inhibits these inflammatory cytokines, the expression of SDF-1 and CXCR4 may be upregulated by HBO through the inhibition of inflammatory cytokines. It is well known that bone marrow (BM)-derived endothelial progenitor cell (EPC) is a key cell involved in neovascularization and homes to peripheral tissue in response to ischemia (26). Endothelial nitric oxide synthase (eNOS) is essential in the BM microenvironment and increased BM NO levels results in the mobilization of EPCs from BM niches into circulation, ultimately allowing their participation in tissue-level vasculogenesis and wound healing (27,28). Induction of hyperoxia via HBO treatment has been shown to increase NO levels in perivascular tissues and BM via a NOS-mediated mechanism, resulting in EPC release from the BM and an increase in EPCs in tissues (29–31). EPCs themselves express and secrete SDF-1 and CXCR4 (32), thus following HBO treatment, the expression of SDF-1 and CXCR4 may be increased. The effects of HBO treatment on the expression of SDF-1 and CXCR4 were supported in the present study.

Ghadge et al(33) indicated that manipulating SDF-1 and its receptor CXCR4 in ischemic cardiac disease is beneficial for neovascularization and tissue repair. Li et al(34) indicated that SDF-1 may induce neovascularization potentially via the SDF-1/CXCR4 axis following traumatic brain injury. Blocking the SDF-1/CXCR4 axis resulted in reduced homing of cells to lesions and decreased MVD after ischemic heart disease (35). These findings suggest that the SDF-1/CXCR4 axis is important in blood vessel growth and development. In the present study, the Pearson’s correlation analysis demonstrated a positive correlation between neovascularization and the expression of SDF-1 and CXCR4 in the skin flaps of rats. There may be various mechanisms involved in the SDF-1/CXCR4 axis governing neovascularization. In addition to its role in recruiting EPCs to ischemic sites (36), SDF-1 directly participates in blood vessel formation. SDF-1/CXCR4 has an angiogenic effect on endothelial cells by inducing cell proliferation, differentiation, sprouting and tube formation in vitro and by preventing apoptosis of EPCs (37). SDF-1 also modulates vascularization of ischemic tissues and tumors by improving the secretion of other angiogenic factors, such as VEGF-A, interleukin-6 (IL-6), IL-8, tissue inhibitor of metalloproteinase-2, and decreasing the production of the anti-angiogenic molecules such as angiostatin (38,39).

In conclusion, the present study established a modified epigastric pedicle skin flap model in rats. Data of this study indicate that HBO treatment promoted neovascularization and increased the survival of skin flaps. In addition, the expression of SDF-1 and CXCR4 was upregulated in the skin flaps of rats treated with HBO. Pearson’s correlation analysis demonstrated a positive correlation between neovascularization and the high expression of SDF-1 and CXCR4. These results suggest that the beneficial effects of HBO treatment in promoting neovascularization may be explained by the upregulation of SDF-1 and CXCR4 expression in the skin flaps of rats.

References