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The induction of endogenous vascular endothelial growth factor (VEGF) production in the skin flap with ischemic injury and the effect of exogenous VEGF on survival of the ischemic skin flap were studied in rats. A dorsal flap model (3×10 cm2) was used in this study. In Part I, biopsies were taken from the flap at 2.5, 5.5, and 8.5 cm distances from the distal edge at 0, 6, 12, and 24 h after the flaps were sutured. Malonyldialdehyde (MDA) and VEGF165 protein level were measured. In Part II, exogenous VEGF (1 μg/ml) was injected subdermally into the flaps in 14 rats before the flaps were replaced. Flaps that received a saline injection were used as the controls. The skin paddle survival was measured on postoperative day five. The results showed that the MDA level in the distal part of the flap significantly increased at 24 h postoperatively when compared to MDA in other parts of the flap. However, VEGF levels in the distal part of the flap significantly decreased when compared to the middle part of the flap. Subdermal injection of exogenous VEGF to the distal area of the flap could significantly improve survival of the distal flap (89% of total skin paddle) when compared to the control, which had a 64% mean percent survival. We conclude that production of endogenous VEGF protein is significantly increased in the skin flap with mild ischemia, but decreased in the flap with severe ischemia. Administration of exogenous VEGF could significantly enhance survival of ischemic flaps.
Partial necrosis of pedicle skin flaps remains a significant problem in plastic and reconstructive surgery. The causes of necrosis are inadequate arterial inflow, insufficient venous outflow, or both.
These factors lead to ischemia that frequently occurs in the portion of the flap most distal to the pedicle. Experimental work has shown that augmenting the vascularity of an ischemic skin flap can significantly improve flap survival.
Angiogenesis, one biological mechanism for the formation of new capillaries from preexisting venules, is fundamental to wound healing and to the maintenance and repair of the vasculature.
The potential of therapeutic agents, including a variety of growth factors, to stimulate the development of angiogenesis in ischemic skin flaps has aroused considerable interest recently.
Vascular endothelium growth factor (VEGF) is an endogenous stimulator of both angiogenesis and vascular permeability. VEGF is expressed in developing blood vessels.
Overexpression of vascular endothelial growth factor in the avian embryo induces hypervascularization and increased vascular permeability without alterations of embryonic pattern formation.
Several experimental studies indicate that an administration of exogenous VEGF can induce regional angiogenesis and improve the survival of random extension of axial pattern skin flaps, as well as accelerate flap prefabrication.
Our previous studies also demonstrated that exogenous VEGF could improve survival of the ischemic rat gracilis muscle flap and the zone IV of the skin paddle in a rat TRAM flap model.
Clarifying how endogenous VEGF induction is regulated in an ischemic skin flap model could provide a key to the understanding of how angiogenesis is controlled by administration of exogenous VEGF in the improvement of survival of the ischemic skin flap.
Free radical mediated tissue injury is an important mechanism of cell damage. Lipid peroxidation is one way in which cells are injured by free radicals. The lipid–radical interactions result in the production of lipid peroxides and their byproducts, which can ultimately lead to the loss of membrane function and integrity.
In many instances, malondialdehyde (MDA) is the most abundant individual aldehyde produced from lipid peroxidation secondary to the attack of free radicals.
In this study, we assayed endogenous VEGF level in a full thickness random pattern skin flap model, and delineated the relationship between endogenous VEGF production, lipoperoxidation, and the effect of exogenous VEGF on survival of the ischemic skin flap in the rat.
1. Materials and methods
Fifty-two male Sprague-Dawley rats weighing between 380 and 420 gm and cared for under the National Research Council's guidelines for the care and use of laboratory animals were used in the study. The rats were anesthetised using pentobarbital administered by intraperitoneal injection (50 mg/kg).
Following induction of general anesthesia, the dorsal regions were shaved, and the animals were placed in a prone position. A caudally based rectangular dorsal flap, measuring 3×10 cm2, was symmetrically raised.
The distal edge of the flap was situated at 4 mm caudal to the lower edges of the scapulae. The flap consisted of skin, panniculus carnosus muscle, and submuscular areolar tissue. In this flap model, two arteries are constantly located at the flap's base and left intact. The flap was then returned to its bed and carefully sutured into place using 4/0 nylon suture. The study was divided into two parts.
1.1 Part I
Twenty-four rats (n=24) were used in Part I. After the flaps were sutured, the animals were divided into four groups of six rats each, according to various time intervals of biopsies immediately, then at 6, 12, and 24 h postoperatively. Two full thickness 4-mm punch biopsies were taken from the flap at 2.5, 5.5, and 8.5 cm distances from the distal edge, respectively (Fig. 1) .
Fig. 1A caudally based rectangle dorsal flap model and punch biopsies taken from the flap with different distances from the distal edge.
The first punch biopsies from the various distances to the edge were homogenised for lipoperoxidation examination, determined by the formation of malonyldialdehyde (MDA) (Lipid Peroxidation Assay Kit, Calbiochem, San Diego, CA). Tissue was weighed and homogenised and the homogenates were diluted in Tris–HCL buffer (pH 7.4) to approximately 10% (w/v). The diluted homogenates were centrifuged at 3000 rpm for 10 min at 4 °C. The supernatant was collected and placed on a vortex for 3–4 s. After 150 μl of 12 N HCl was added, the samples were cooled on ice and the absorbance was read at 540 nm in a spectrophotometer. The amount of MDA was calculated using an extinction coefficient (1.56×105/M/cm) and expressed as nanomoles per 10 mg wet weight.
1.3 VEGF level determination
Tissue samples from the second punches were homogenised in 400 μl PBS (pH 7.2). The homogenates were centrifuged at 15,000 rpm for 30 min at 4 °C. The supernatant was collected and stored at −80 °C until used. VEGF165 was determined with an enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN). Standards or samples (50 μl) were pipetted into each antibody-coated well containing 50 μl assay diluent and incubated for 2 h at room temperature. The wells were washed five times with wash buffer, and then 100 μl VEGF conjugate was added to each well. The samples were again incubated for 2 h at room temperature. After washing five times, 100 μl substrate solution was added. Samples were incubated for 30 min at room temperature. The reaction was stopped by adding 100 μl stop solution. Optical density was read in a plate reader (Bio-Tec Instruments, Inc., Winnoski, VT) at the wavelength of 450 nm with a reference at 570 nm. The tissue sample concentration was calculated from the standard curve and normalised by the weight of the skin.
Mean and standard deviation (SD) of MDA and VEGF levels of proximal, middle, and distal skin at each time period were statistically compared by using one way ANOVA, followed by Turky's comparison. The significant level was assumed at p<0.05.
1.4 Part II
The 165 amino acid isoform of recombinant human VEGF (Genentech Inc., South San Francisco, CA) suspended in phosphate-buffered saline was used in this study. The remaining 28 rats were divided into two groups, experimental (n=14) and control (n=14) groups, based on the given treatment protocol.
In the experimental group, before the flap was replaced, 1 ml of exogenous VEGF (1 μg/ml) was injected subdermally into the distal 3×4 cm2 area of the flap, by administering 0.1 ml of VEGF at 10 equally distributed locations on the thick fascial layer composing the undersurface of the flap. In the control group, the flap received the same amount of saline after elevation of the flap.
The animals were returned to their individual cages for a period of 5 days, after which they were reanesthetised as described above. The survival area of the skin paddle was grossly determined based on appearance, colour, and texture. It was then marked on a template. With the aid of an M2 Image Analysis System (Imaging Research Inc., St Catharines, Ontario, Canada), the template was photographed by a high-resolution video camera and scanned on a computer. The animals were then sacrificed by an overdose of pentobarbital. The skin paddle was placed in 10% formalin. The tissue samples from the necrotic portions and survival areas at 1 cm proximal to the necrosis-survival margin were sectioned with haematoxylin-eosin staining for histological analysis. The data from different groups were compared using analysis of variance (ANOVA) and Student's t-test. Statistical significance was assumed at p<0.05.
2. Results
2.1 Lipoperoxidation
Fig. 2 depicts MDA changes over time at different biopsy sites. There were no significant differences in MDA levels between different locations on the flaps in the first 12 h after surgery (p>0.05). However, the MDA level in the distal part of the flaps increased (2.66±0.32 nmol/10 mg weight, mean±SD) at 24 h postoperatively, which was significantly higher than MDA at the same location at 12 h (1.23±0.37 nmol/10 mg) (p<0.05). The MDA level in the distal portion of the flaps was also significantly higher than MDA in the middle and proximal portions of the flaps (1.79±0.31 nmol/10 mg and 1.32±0.28 nmol/10 mg) at 24 h postoperatively (p<0.01).
Fig. 2MDA changes over time at different biopsy sites (n=6 for each time period).
The values of VEGF protein, from different parts of the flap at each time interval, are shown in Fig. 3. The VEGF level in the normal tissue was low during surgery (0–0.1 pg/mg tissue weight), and slightly increased at 6 h after the flap was raised. At 12 h postoperatively, VEGF level in the mid portion of the flap significantly increased (0.62±0.17 pg/mg, mean±SEM) when compared to the proximal flap (0.23±0.05 pg/mg) (p<0.01). In contrast, VEGF level in the distal portion did not increase (0.34±0.03 pg/mg). At 24 h after the flaps were raised, VEGF level in the distal portion of the flaps (0.52±0.07 pg/mg) was significantly lower than that in the middle portion of the flaps (0.94±0.32 pg/mg) (p<0.01). The VEGF level in the proximal portions without ischemia remained low (0.39±0.12 pg/mg).
Fig. 3The value of VEGF protein from different parts of the flap at each time interval (n=6 for each time period).
At day 5 postoperatively, the regions of survival and necrosis were clearly demarcated in every flap in Group II. The surviving skin appeared pink–white, tender, normal in its texture, and bled when cut with a scalpel. In contrast, the necrotic skin was black, rigid, and did not bleed when cut. The results of flap survival are summarised in Table 1. The experimental group treated with subdermal exogenous VEGF injection demonstrated a mean and SD of the viable area of 26.7±1.8 cm2 (89% of the total skin paddle) (Fig. 4) , which was significantly greater compared to the control (19.2±1.2 cm2, 64% of the total skin paddle, Fig. 5) (p<0.05).
Table 1Mean area and percent survival of flaps from rats treated with VEGF and control rats receiving no treatment (results from postoperative day 5)
Group
Mean area of survival in cm2 (total flap area=30 cm2)
Mean percent survival (%)
Subdermal VEGF (n=14)
26.7±1.8
89
Control (no treatment) (n=14)
19.2±1.2
64
Note: values following ‘±’ indicate standard deviation (SD).
The sections were taken from both necrotic and surviving regions. Necrotic sections were similar in both experimental and control groups, and revealed evidence of acute inflammation with infiltration of monocytes and neutrophils. Myonecrosis was evident in about 90% of the muscle layer in these sections. In the flaps from VEGF-treated animals, sections taken from surviving regions close to the necrosis-survival margin revealed a consistent pattern of exaggerated amounts of granulation tissue and neovascularisation (Fig. 6) . This was most apparent deep to the muscle layer, with evidence of granulation tissue invasion into this layer. The sprouting of new vessels appeared to be primarily capillary in origin. Sections taken from viable regions of flaps from the same areas in the control group failed to show the extent of granulation tissue and angiogenesis.
Fig. 6Neovascularisation in the flap with VEGF treatment.
to study skin flap necrosis and its prevention. The design of the flap has since been modified several times. The survival pattern of this flap model has been demonstrated to be different than that of the skin graft.
which is perfused by two constant sacral axial vessels. Inadequate blood supply to the distal part of the flap creates varying degrees of ischemia, which allows for the investigation of flap physiology, angiogenesis, and augmentation of flap survival.
MDA levels are indicative of lipoperoxidation, a process closely related to free radical mechanisms. It is believed that free radicals are important mediators of destruction during ischemic injury. They can participate in chain reactions and cause peroxidation of cellular and intracellular membranes and intracellular proteins resulting in irreversible cell injury.
The lipoperoxidation was greatly elevated at 24 h in the distal part of the flap in our study. This finding confirms previous reports of ischemia-reperfusion injury in this flap model.
VEGF induces angiogenesis and endothelial cell proliferation, and plays an important role in regulating vasculogenesis. VEGF is a heparin-binding glycoprotein that is secreted as a homodimer of 45 kDa
The vascular endothelial growth factor (VEGF) isoforms: differential deposition into the subepithelial extracellular matrix and bioactivity of extracellular matrix-bound VEGF.
Overexpression of vascular endothelial growth factor in the avian embryo induces hypervascularization and increased vascular permeability without alterations of embryonic pattern formation.
However, the precise role of endogenous and exogenous VEGF in survival of the skin flap with ischemic injury is not documented. Previous studies indicate that an administration of exogenous VEGF can improve survival of random skin flaps.
To understand endogenous VEGF expression in the ischemic skin flap would provide the evidence needed for a clinical use of exogenous VEGF to enhance angiogenesis and ischemic flap survival.
The VEGF family is known to include VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E and placental growth factor (PIGF).
VEGF-A is the most predominant protein. There are several splice variants of VEGF-A. VEGF165 is the major isoform in all cells and tissues. In our study, the linear relationship between the VEGF165 protein level and the degree, as well as time of ischemia was examined in the skin flap. The production of endogenous VEGF was found to be significantly increased in response to the mild ischemia in the middle portion of the flap. Survival of this part of the flap may be attributed to the effect of endogenous VEGF on inducing endothelial cell proliferation and reducing the endothelial dysfunction, which follows free radicals related ischemia-reperfusion injury.
Overexpression of vascular endothelial growth factor in the avian embryo induces hypervascularization and increased vascular permeability without alterations of embryonic pattern formation.
However, the endogenous VEGF level in the severely ischemic area of the distal flap was low. It could be presumed that VEGF protein synthesis is depressed by low oxygen supply and free radicals formation. In evaluation of flap survival, most of the proximal and middle portions of the flap were viable, consisting of approximately 64% of the total skin paddle. However, the most distal part of the flap was noted to be necrotic.
Administration of exogenous VEGF to the flap with severe ischemia could significantly enhance survival of this portion of the flap. In our study, the survival rate was improved to 89% when the flap received subdermal VEGF injection. Exaggerated amounts of granulation tissue formation and neovascularisation could be induced in the distal part of the skin flap by exogenous VEGF. The maintenance of normal endothelial function and formation of new vessels by exogenous VEGF appeared to be the major mechanism to augment survival of this part of the flap.
In conclusion, our results suggest that induction of VEGF protein is significantly up-regulated by mild ischemia in the skin flap. However, the VEGF level is not elevated when the flap is in severe ischemia. Administration of exogenous VEGF could significantly enhance viability of the ischemic flap and these data provide evidence for clinical administration of exogenous VEGF to improve ischemic flap survival.
Overexpression of vascular endothelial growth factor in the avian embryo induces hypervascularization and increased vascular permeability without alterations of embryonic pattern formation.
The vascular endothelial growth factor (VEGF) isoforms: differential deposition into the subepithelial extracellular matrix and bioactivity of extracellular matrix-bound VEGF.
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