Enhancement of epigastric skin flap survival by adenovirus-mediated VEGF gene therapy

A novel approach to treat ischemic tissues by using gene therapy has recently been introduced on the basis of the angiogenic potential of certain growth factors. The authors investigated the effect of adenovirus-mediated gene therapy with vascular endothelial growth factor (VEGF) delivered into the subdermal space to treat compromised skin flaps. For this purpose, the epigastric skin flap model in rats, based solely on the right inferior epigastric vessels, was used. Thirty male Sprague-Dawley rats were divided into five groups of six rats each. Viral transfection with 108 plaque-forming units was performed 2 days before the epigastric flap elevation. Rats received subdermal injections of adenovirus encoding VEGF (Ad-VEGF) or green fluorescent protein (Ad-GFP) as treatment control. Another set of animals (n = 6) received no injections and were designated as control. To determine whether site of injection had an impact on flap viability, injections were given into the predicted local ischemic area (Ad-VEGF local, n = 6; Ad-GFP local, n = 6) and into the midline of the flap (Ad-VEGF midline, n = 6; Ad-GFP midline, n = 6). A flap measuring 8 x 8 cm was outlined on the abdominal skin extending from the xiphoid process proximally and the pubic region distally, to the anterior axillary lines bilaterally. Then, the epigastric flap was elevated as an island on the right inferior epigastric vessels and sutured back to its bed. Flap viability was evaluated at 7 and 14 days after the first operation. The epigastric flaps were scanned to the computer and areas of hypoxic and/or necrotic zones relative to total flap surface area were measured and expressed as percentages by using Image Pro Plus software. Specimens were taken for histologic evaluation at day 14 before the animals were killed. Combined area of necrotic and hypoxic zones as well as necrotic zone were decreased to 9.7 +/- 1.4 percent and 1.4 +/- 0.9 percent in Ad-VEGF local, and 11.8 +/- 1.9 percent and 3.5 +/- 1.64 percent in Ad-VEGF midline compared with the control and Ad-GFP treatment groups (control, 23 +/- 3.6 percent and 20.1 +/- 3.3 percent; Ad-GFP local, 24.8 +/- 4.8 percent and 16.2 +/- 5.9 percent; and Ad-GFP midline, 23.4 +/- 6.9 percent and 19.5 +/- 7.7 percent; p < 0.05). Histologic evaluation by light microscopy failed to demonstrate any quantitative difference in vascularity of skin flaps between the treatment groups. In this study, the authors demonstrated that adenovirus-mediated gene therapy using VEGF enhanced epigastric skin flap survival, as confirmed by the significant reduction in combined area of necrotic and hypoxic zones of the flap. Compared with the control, both local and midline subdermal injections of Ad-VEGF showed improvement in overall flap survival by 57.9 and 48.7 percent, respectively. The results of this study raise the possibility of using adenovirus-mediated therapeutic angiogenesis for safer flap surgery in high-risk patients.     

Vascular endothelial growth factor expression in pig latissimus dorsi myocutaneous flaps after ischemia reperfusion injury

Exogenous administration of vascular endothelial growth factor (VEGF) improves long-term viability of myocutaneous flaps. However, endogenous expression of this substance in flaps following ischemia-reperfusion injury has not been reported previously. Endogenous production of VEGF was measured in myocutaneous pig latissimus dorsi flaps after ischemia-reperfusion injury. Latissimus dorsi myocutaneous flaps (15 x 10 cm) were simultaneously elevated bilaterally in six Yorkshire-type male pigs (25 kg). Before elevation, three flap zones (5 x 10 cm) were marked according to their distance from the vascular pedicle. After isolation of the vascular pedicle, ischemia-reperfusion injury was induced in one flap by occlusion of the thoracodorsal artery and vein for 4 hours, followed by 2 hours of reperfusion. The contralateral flap served as a control. Perfusion in each zone was monitored by laser Doppler flowmetry at baseline, during ischemia, and during reperfusion. At the end of the protocol, skin and muscle biopsies of each flap zone and adjacent tissues were obtained for later determination of VEGF protein levels. VEGF concentrations were quantified using the Quantikine human VEGF immunoassay. Skin perfusion was similar among all flap zones before surgery. Flow fell in all flaps immediately after flap elevation. After 4 hours of ischemia, blood flow in the ischemic flaps was significantly decreased (p < 0.05) compared with nonischemic control flaps. After 2 hours of reperfusion, flow in ischemic flap skin recovered to levels similar to those in control flaps. VEGF protein concentrations in muscle tissue exceeded concentrations in skin and decreased from zones 2 to 3 in control and ischemic flaps. No significant differences in VEGF concentrations between ischemic and control muscle zones were observed. However, the concentration of VEGF in all muscle zones was significantly higher (p < 0.05) than muscle adjacent to the flap. Concentrations in skin zones 1 and 2 were significantly higher (p < 0.05) in ischemic flaps than in control flaps, but levels in zone 3 (most ischemic flaps) showed no significant difference.     

Early and late effects of ischemic preconditioning on microcirculation of skeletal muscle flaps

The present study was designed to investigate the early and late effects of ischemic preconditioning on muscle flap perfusion and reperfusion-induced skeletal muscle damage. Thirty-six Sprague-Dawley rats were divided into six experimental groups of six animals each. The cremaster muscle flap model and the intravital microscopy system were used to observe microcirculatory changes associated with ischemia-reperfusion injury and ischemic preconditioning. In groups 1, 2, and 3, microcirculatory measurements were taken on the same day; however, in groups 4, 5, and 6, measurements were taken a day after surgery. Group 1 served as a control. The cremaster muscle was prepared as a tube flap, subjected to an hour of perfusion without ischemia. In group 2 (ischemic preconditioning + ischemia group), the cremaster muscle tube flap was subjected to 30 minutes of ischemia and 30 minutes of reperfusion, followed by 4 hours of total ischemia. In group 3 (ischemia alone), the flap was submitted to 4 hours of ischemia alone. In group 4 (control), the cremaster muscle flaps were dissected out, preserved in the subcutaneous tunnel, and submitted to 24 hours of perfusion only. In group 5 (ischemic preconditioning + 24 hours of perfusion + 4 hours of ischemia), the ischemic preconditioning protocol was followed by 24 hours of perfusion and 4 hours of ischemia. In group 6 (24 hours of perfusion + ischemia), the same protocol was used as in group 5 without ischemic preconditioning. Functional capillary perfusion, and the diameters of the arterioles of the first, second, and third order were significantly increased in the ischemic preconditioning group during the early period, but not after 24 hours of perfusion. No differences in the red blood cell velocities of arterioles of the first, second, or third order were found in either the early-effect or late-effect groups. The numbers of rolling, adhering, and transmigrating leukocytes, however, were significantly lower in the ischemic preconditioning group at both early and late follow-up. Ischemic preconditioning of the skeletal muscle flap has both an early and a late protective effect against reperfusion injury. Ischemic preconditioning at the early interval significantly improves muscle flow hemodynamics of the flap and attenuates leukocyte-mediated reperfusion injury. After 24 hours of reperfusion, however, ischemic preconditioning failed to improve the flow hemodynamics of the flap, yet it still protected the skeletal muscle flap from leukocyte-mediated reperfusion injury.     

Blockade of platelet endothelial cell adhesion molecule-1 (PECAM-1) protects against ischemia-reperfusion injury in muscle flaps at microcirculatory level

Several lines of evidence show that platelet endothelial cell adhesion molecule-1 (PECAM-1), a component of endothelial cell junctions, is required for leukocyte transmigration through endothelial cell monolayers. Polymorphonuclear leukocytes play an important role in ischemia-reperfusion injury. We sought to determine whether administering an anti-PECAM-1 antibody would prevent or attenuate ischemia-reperfusion injury in a rat cremaster muscle flap injury model. Eighteen male Sprague-Dawley rats were divided into three groups. Group I (control): Cremaster muscle island flaps were dissected for baseline measurements of eight indicators: numbers of rolling, sticking, and transmigrating neutrophils, numbers of rolling and sticking lymphocytes, number of perfused capillaries, endothelial edema, and vessel permeability. Group II: The prepared cremaster flap was subjected to 4 hours of ischemia and 24 hours of reperfusion. Group III: The muscle flap was subjected to ischemia and reperfusion as in group II, and anti-PECAM-1 antibodies (1 mg/kg) were injected subcutaneously 15 minutes before reperfusion. Blood vessels were observed in vivo under an intravital microscopy system. Microvascular permeability was made visible with injected fluorescein isothiocyanate-labeled albumin and evaluated with Kontron Elektronik computer software. The ischemia-reperfusion-alone group (group II) presented a 225-percent increase in the activation of sticking leukocytes (2.4 +/- 0.4 to 7.8 +/- 0.8, p < 0.05) (p < 0.01). This leukocyte activation was reduced by 83 percent following anti-PECAM-1 monoclonal antibody treatment (1.3 +/- 0.5 per 100 microm) (p < 0.01). At 24 hours, endothelial injury in group II was confirmed by a 4-fold increase in the number of transmigrating leukocytes into the interstitial space (7.6 +/- 1.2 per field versus 1.9 +/- 0.4 per field in controls). This phenomenon was reduced by 85 percent following anti-PECAM-1 monoclonal antibody treatment (1.1 +/- 0.2 per field) (p < 0.01). Analysis showed that the number of flowing capillaries was 67 percent lower in group II (6.8 +/- 0.3 to 2.2 +/- 0.7, p < 0.01). Anti-PECAM-1 antibody treatment caused a 2.5-fold increase in this number (5.6 +/- 0.5, p < 0.01). Microcirculatory permeability index showed a 180-percent increase in group II (p < 0.05) when compared with baseline values. This increased albumin leakage was effectively attenuated by antibody treatment (p < 0.05). Blocking the action of PECAM-1 in vivo by administering monoclonal antibodies significantly attenuated ischemia-reperfusion injury, presumably by inhibiting transendothelial migration of neutrophils and by increasing capillary perfusion at a muscle flap microcirculatory level.     

Improved perfusion after subcritical ischemia in muscle flaps treated with vascular endothelial growth factor

Vascular endothelial growth factor (VEGF), a potent endothelial mitogen, is secreted in ischemic tissue and plays a pivotal role in angiogenesis. We studied whether VEGF administered to a rat muscle flap at the time of ischemia induction would increase microcirculatory flow to the flap. The cremaster muscle flap was isolated on its neurovascular pedicle. Ischemia was induced by clamping the vascular pedicle, and 0.2 ml of either VEGF (0.1 microg) or vehicle (phosphate-buffered saline) was immediately infused into the muscle. After 4 or 6 hours, the clamps were released, and the cremaster was placed in a pocket in the medial thigh for 24 hours. The muscle was then dissected, and microcirculatory measurements were made under intravital microscopy. Six animals were used in each of the four groups. All flaps exposed to 6 hours of ischemia, the duration considered to be critical ischemia, had no significant microcirculatory flow, regardless of treatment with VEGF. In the 4-hour ischemia group, or subcritical ischemia group, red blood cell velocity in arterioles was 14 mm/sec in muscles treated with VEGF and 9 mm/sec in controls (p = 0.02), and capillary flow was 7 per high-power field in muscles treated with VEGF versus 2 per high-power field in controls (p = 0.0005). Thus, VEGF did not alter microcirculatory flow in a muscle flap exposed to critical ischemia, but it did enhance flow to a flap exposed to subcritical ischemia.     

Muscle flaps' triphasic microcirculatory response to sympathectomy and denervation.

Whether sympathectomy and somatic denervation in muscle flaps increased microcirculatory flow in the short or long term, thus producing an effect similar to the delay phenomenon, which increases survival in transferred skin flaps, was determined. The rat cremaster muscle flap model was used for in vivo microscopy. In the left cremasters of 30 Sprague-Dawley rats, the genitofemoral nerve was divided and the proximal vessels were stripped of their adventitia. The muscle was not elevated. In each rat, the contralateral cremaster served as the control. The rats were assigned to one of five groups: no delay before observation, a 24-hour delay, a 48-hour delay, a 7-day delay, or a 14-day delay. After the delay, red blood cell velocity, vessel diameters, number of functional capillaries, and leukocyte-endothelial interactions were measured. Microvessel response to topical vasoactive substances was measured. Immediately after denervation, red blood cell velocity increased transiently (71 percent; p = 0.006). Main arterioles dilated (20 percent; p = 0.02) at 24 hours, and capillary perfusion increased 36 percent (p = 0.001) at 2 weeks. The microvessels had hyperactive responses to all vasoactive agents 2 weeks after denervation. These findings indicate that proximal sympathectomy with somatic denervation leads to a triphasic, dynamic response in the peripheral microcirculation of the cremaster muscle flap. An initial acute hyperadrenergic phase was followed by a nonadrenergic phase, with significant vasodilatation, and a sensitized phase, with increased capillary perfusion and hyperresponsiveness to vasoactive substances. This study shows that with minimal access to the cremaster muscle flap neurovascular pedicle and without changing the blood supply to the flap, significant hemodynamic improvements can be made in the peripheral microcirculation.     

IRES/polyA-promoter介导的Ang-1与VEGF165双基因表达效率的对比分析

目的:构建IRES及polyA-promoter介导人血管生成素-1(Angiogenin-1,Ang-1)和人血管内皮生长因子165(vascular endothelial growth factor165,VEGF165)双基因共表达的腺病毒载体,比较IRES与polyA-promoter不同表达模式及其对位于二者前后基因的表达效率和诱导兔角膜新生血管的形成功能,为今后构建双基因或多基因高效共表达载体提供实验依据。方法:以pAdTrack-CMV-Ang-1-IRES-VEGF165质粒为模板,PCR扩增人VEGF165及Ang-1基因片段,分别将其亚克隆至改建的 pAdTrack-CMV-PolyA-promoter及pAdTrack-CMV-IRES转移质粒中,构建pTrack-CMV-Ang-1-polyA-promoter-VEGF165、pTrack-CMV- VEGF165-polyA-promoter-Ang-1、pTrack-CMV-VEGF165-IRES-Ang-1基因重组转移质粒,再与腺病毒骨架质粒pAdeasy-1在BJ5183细菌中同源重组,然后经PacI线性化后转染QBI-293A人胚肾成纤维细胞(简称293A细胞),收获腺病毒重组病毒子Ad-Ang-1-polyA-promoter-VEGF165及Ad-VEGF165-polyA-promoter-Ang-1,Ad-VEGF165-IRES-Ang-1,RT-PCR检测Ang-1和VEGF165在QBI-293A细胞中的转录,ELISA法分别检测不同腺病毒载体目的基因的表达量,比较分析Ang-1与VEGF165基因在IRES和polyA-promoter介导的不同腺病毒表达载体中的表达能力,及在同一腺病毒表达载体中前后不同位置的表达效率。并进一步于兔角膜缘注射Ad-Ang-1-polyA-promoter-VEGF165,Ad-VEGF165-polyA-promoter-Ang-1,Ad-VEGF165-IRES-Ang-1,Ad-Ang-1-IRES-VEGF165,检测角膜新生血管的面积,并比较其诱导血管形成能力的差异。结果:测序显示Ang-1和VEGF165序列正确,不同重组腺病毒载体均获得成功包装,病毒效价可达2~5×10¹⁰pfu/ml,RT-PCR检测Ang-1和VEGF165均能有效转录,ELISA法检测结果表明Ang-1、VEGF165基因不仅均能在细胞中有效表达,而且IRES介导的Ang-1及VEGF165基因,无论在IRES上游或下游,其表达量均低于polyA-promoter相同位置的Ang-1及VEGF165基因表达量,大约降低60%~70%左右,同时Ang-1/VEGF165在同一载体上、下游不同位置,其下游基因的表达量均明显低于上游基因表达量,大约降低30%~40%左右。角膜血管形成动物实验的结果表明Ad-VEGF165-PolyA-promoter-Ang-1及Ad-VEGF165-IRES-Ang-1诱导角膜新生血管形成面积和血管密度的能力相对较强,且前者比后者效果更为显著。结论:在腺病毒表达载体中,由IRES/polyA-promoter介导的Ang-1与VEGF165双基因均能在细胞中成功表达,并具血管诱导性,但polyA-promoter比IRES介导的双基因表达效率高,诱导血管形成能力强;同时两者下游基因的表达量及血管诱导性能均明显低于上游基因。     

Non‐viral VEGF165 gene therapy – magnetofection of acoustically active magnetic lipospheres (‘magnetobubbles’) increases tissue survival in an oversized skin flap model

Adenoviral transduction of the VEGF gene in an oversized skin flap increases flap survival and perfusion. In this study, we investigated the potential of magnetofection of magnetic lipospheres containing VEGF₁₆₅‐cDNA on survival and perfusion of ischemic skin flaps and evaluated the method with respect to the significance of applied magnetic field and ultrasound. We prepared perfluoropropane‐filled magnetic lipospheres (‘magnetobubbles’) from Tween60‐coated magnetic nanoparticles, Metafectene, soybean‐oil and cDNA and studied the effect in an oversized random‐pattern‐flap model in the rats (n= 46). VEGF‐cDNA‐magnetobubbles were administered under a magnetic field with simultaneously applied ultrasound, under magnetic field alone and with applied ultrasound alone. Therapy was conducted 7 days pre‐operative. Flap survival and necrosis were measured 7 days post‐operatively. Flap perfusion, VEGF‐protein concentration in target and surrounding tissue, formation and appearance of new vessels were analysed additionally. Magnetofection with VEGF‐cDNA‐magnetobubbles presented an increased flap survival of 50% and increased flap perfusion (P < 0.05). Without ultrasound and without magnetic field, the effect is weakened. VEGF concentration in target tissue was elevated (P < 0.05), while underlying muscle was not affected. Our results demonstrate the successful VEGF gene therapy by means of magnetobubble magnetofection. Here, the method of magnetofection of magnetic lipospheres is equally efficient as adenoviral transduction, but has a presumable superior safety profile.     

Vasodilator effect and mechanism of action of vascular endothelial growth factor in skin vasculature

Various laboratories have reported that local subcutaneous or subdermal injection of VEGF(165) at the time of surgery effectively attenuated ischemic necrosis in rat skin flaps, but the mechanism was not studied and enhanced angiogenesis was implicated. In the present study, we used the clinically relevant isolated perfused 6 x 16-cm pig buttock skin flap model to 1) test our hypothesis that VEGF(165) is a potent vasodilator and acute VEGF(165) treatment increases skin perfusion; and 2) investigate the mechanism of VEGF(165)-induced skin vasorelaxation. We observed that VEGF(165) (5 x 10(-16)-5 x 10(-11) M) elicited a concentration-dependent decrease in perfusion pressure (i.e., vasorelaxation) in skin flaps preconstricted with a submaximal concentration of norepinephrine (NE), endothelin-1, or U-46619. The VEGF(165)-induced skin vasorelaxation was confirmed using a dermofluorometry technique for assessment of skin perfusion. The vasorelaxation potency of VEGF(165) in NE-preconstricted skin flaps (pD(2) = 13.57 +/- 0.31) was higher (P < 0.05) than that of acetylcholine (pD(2) = 7.08 +/- 0.24). Human placental factor, a specific VEGF receptor-1 agonist, did not elicit any vasorelaxation effect. However, a specific antibody to VEGF receptor-2 (1 microg/ml) or a specific VEGF receptor-2 inhibitor (5 x 10(-6) M SU-1498) blocked the vasorelaxation effect of VEGF(165) in NE-preconstricted skin flaps. These observations indicate that the potent vasorelaxation effect of VEGF(165) in the skin vasculature is initiated by the activation of VEGF receptor-2. Furthermore, using pharmacological probes, we observed that the postreceptor signaling pathways of VEGF(165)-induced skin vasorelaxation involved activation of phospholipase C and protein kinase C, an increase in inositol 1,4,5-trisphosphate activity, release of the intra-cellular Ca(2+) store, and synthesis/release of endothelial nitric oxide, which predominantly triggered the effector mechanism of VEGF(165)-induced vasorelaxation. This information provides, for the first time, an important insight into the mechanism of VEGF(165) protein or gene therapy in the prevention/treatment of ischemia in skin flap surgery and skin ischemic diseases.     

Improved dorsal random-pattern skin flap survival in rats with a topically applied combination of nonivamide and nicoboxil

The effects of a topically applied combination of nonivamide and nicoboxil in improving skin perfusion and preventing distal flap necrosis were tested in a random-pattern dorsal skin flap model. Forty male Wistar rats were randomized into two groups (n = 20), and a standardized dorsal random-pattern skin flap was raised on each rat. Animals in the experimental group were treated with the topically applied drug combination four times per day for 6 days, whereas in the control group only a placebo ointment was applied each time. Skin flap viability was evaluated on day 7, and the extent of skin flap necrosis was compared between the two groups. The topically applied combination of nonivamide and nicoboxil resulted in a statistically significant decrease in skin flap necrosis, compared with the control group (mean percentage of skin flap necrosis in the nonivamide/nicoboxil-treated group, 22.6 +/- 6.0 percent; control group, 36.8 +/- 4.3 percent; p< 0.05). The topical combination of nonivamide and nicoboxil was effective in reducing ischemic necrosis in failing random-pattern skin flaps in this rat model. The results of this study suggest that such a topical drug application might have significant effects in the reduction of ischemic necrosis in the distal parts of skin flaps, and this treatment might also have applications as prophylactic therapy for risky skin flaps.     

Efficacy of combination gene therapy with multiple growth factor cDNAs to enhance skin flap survival in a rat model

The objective of this study was to investigate the efficacy of combination gene therapy with multiple angiogenic growth factor cDNAs to enhance survival of ischemic skin flaps in a rat model. Sixty Sprague-Dawley rats were divided into six groups. Varying combinations of VEGF165, PDGF-B, and bFGF-plasmids were injected to prefabricate the flaps. Random skin flaps were raised on the dorsal aspect of rats following prefabrication with growth factor cDNAs. Flap viability was determined by measurement of percentage area of survival. The efficacy of gene therapy was evaluated by flap survival and neovascularization of representative histologic sections stained immunohistologically. The VEGF165 plus bFGF cDNAs enhanced the viability of the flap and neovascularization most effectively; the flap survival area was 64.3 +/- 8.7% after transfer of these two growth factor genes. Addition of PDGF-B cDNA is deleterious to the effects of combined VEGF165 and bFGF, leading to a significant decrease in flap viability (44.9 +/- 2.7%). Viability of the flaps with combined VEGF165 and bFGF cDNA transfer was significantly greater than that of the flaps with VEGF165 transfer alone (57.6 +/- 5.2%) or sham plasmid control (52.3 +/- 5.0%). Combined transfer of VEGF165 and bFGF cDNA is the most effective combination of multiple growth factor genes to improve flap viability in this model. Simultaneous transfer of three growth factor genes (VEGF165, PDGF-B, and bFGF) is deleterious to flap survival, at least for the ratio of lipofectin:transgene employed.     

Acute local subcutaneous VEGF165 injection for augmentation of skin flap viability: efficacy and mechanism

Distal skin ischemic necrosis is a common complication in skin flap surgery. The pathogenesis of skin flap ischemic necrosis is unclear, and there is no clinical treatment available. Here, we used the 4 x 10 cm rat dorsal skin flap model to test our hypothesis that subcutaneous injection of vascular endothelial growth factor 165 (VEGF165) in skin flaps at the time of surgery is effective in augmentation of skin flap viability, which is associated with an increase in nitric oxide (NO) production, and the mechanism involves 1) an increase in skin flap blood flow in the early stage after surgery and 2) enhanced angiogenesis subsequently to sustain increased skin flap blood flow and viability. We observed that subcutaneous injection of VEGF165 in skin flaps at the time of surgery increased skin flap viability in a dose-dependent manner. Subcutaneous injection of VEGF165 at the dose of 2 microg/flap increased skin flap viability by 28% (P < 0.05; n = 8). Over 80% of this effect was blocked by intramuscular injection of the NO synthase (NOS) inhibitor Nomega-nitro-L-arginine (13 mg/kg) 45 min before surgery (P < 0.05; n = 8). The VEGF165 treatment also increased skin flap blood flow (2.68 +/- 0.63 ml x min(-1) x 100 g(-1)) compared with the control (1.26 +/- 0.10 ml x min(-1) x 100 g(-1); P < 0.05, n = 6) assessed 6 h postoperatively. There was no change in skin flap capillary density at this time point. VEGF165-induced increase in capillary density (32.2 +/- 1.1 capillaries/mm2; P < 0.05, n = 7) compared with control (24.6 +/- 1.4 capillaries/mm2) was seen 7 days postoperatively. There was also evidence to indicate that VEGF165-induced NO production in skin flaps was stimulated by activation of NOS activity followed by upregulation of NOS protein expression. These observations support our hypothesis and for the first time provide an important insight into the mechanism of acute local VEGF165 protein therapy in mitigation of skin flap ischemic necrosis.     

AdVEGF165 gene transfer increases survival in overdimensioned skin flaps

BACKGROUND: Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis. VEGF A also plays an important role in wound healing of the skin by promoting angiogenesis and by stimulating blood vessel growth. Therefore we tested the hypothesis that flap survival could be increased by the preoperative injection of AdVEGF(165). METHODS: We studied the effect of AdVEGF(165) in an overdimensioned ischemic random-pattern-flap model in the rat (n = 50) with a length-to-width ratio of 4 : 1. VEGF cDNA was administered in two concentrations of 5 x 10(8) plaque-forming units (pfU) and 1 x 10(9) pfU using a recombinant adenoviral vector. Recombinant virus was injected subdermally 7, 3 or 0 days prior to flap harvest for the lower concentration and 7 days prior for the higher concentration. Flap survival and necrosis were observed at day 7, the day the animals were sacrificed. RESULTS: Adenoviral gene transfer with VEGF(165) 3 and 7 days before flap harvest showed a significantly increased flap survival of 50% together with a significantly reduced necrosis (p < 0.01). Injection using a titer of 1 x 10(9) pfU 7 days prior to surgery increased flap survival even more, though failing to reach statistical significance compared to the lower concentration. VEGF protein concentration in the injected skin was significantly higher than in controls (p < 0.01). Flap perfusion was increased as well, demonstrated by indocyanine green (ICG) fluoroscopy (p < 0.001). CONCLUSIONS: Our results confirm the important role of VEGF(165) on angiogenesis in ischemic flaps. Indeed by injecting VEGF(165) at 3 to 7 days preoperatively in a concentration of 1 x 10(9) pfU our data show that length-to-width ratio for random-pattern-flaps could be increased from 2 : 1 to 3 : 1 and therefore may allow a wider range of applications of this simple flap technique.     

Effects of 8-Gy radiation on the microcirculation of muscle flaps in the rat.

Combination of radical excision and radiation has been used as a treatment modality for cancer patients. As a result, in reconstructive surgery there is often a need to harvest flaps in the vicinity of previously irradiated tissues. Radiation has been shown to cause progressive injury to the macrocirculation and microcirculation, often jeopardizing flap survival. The purpose of this study was to examine whether radiation significantly affects the sequence of leukocyte-endothelial interactions or the hemodynamics of the muscle flap in both acute and chronic situations. Male Sprague-Dawley rats (n = 42) were divided into seven groups of six rats each. Rats in group I were not irradiated. Groups II through VII received 8-Gy radiation to the right groin and scrotum. Groups II, III, and IV were examined at 4, 24 and 72 hours, respectively, and groups V, VI, and VII were examined at 1, 2 and 12 weeks. For intravital microscopy, the cremaster muscle was dissected on its neurovascular pedicle. Vessel diameters and red blood cell velocities were measured in the central cremasteric branches and branch arterioles. Capillary perfusion was evaluated in 27 visual fields of each flap. Leukocyte-endothelial interactions were evaluated by numbers of rolling, adhering, and transmigrating leukocytes in post-capillary venules. In the same postcapillary venule, we measured the endothelial edema index (constriction index). The hemodynamics of irradiated flaps did not differ significantly from those of controls. Diameter and red blood cell velocity were increased in the first- and second-order arterioles and were highest at 72 hours and 1 week. After irradiation, third-order arterioles were constricted. Radiation reduced capillary perfusion by 4.3, percent. None of the differences were statistically significant. Neither leukocyte behavior nor the constriction indices differed among the groups. In conclusion, low-dose radiation of 8 Gy does not affect hemodynamics or leukocyte-endothelial interactions of muscle flaps in the rat. Muscle tissue with intact microvasculature can be harvested for reconstructive procedures after low-dose radiation.     

Effect of vascular delay on viability, vasculature, and perfusion of muscle flaps in the rabbit

The delay procedure is known to augment pedicled skin or muscle flap survival. In this study, we set out to investigate the effectiveness of vascular delay in two rabbit muscle flap models. In each of the muscle flap models, a delay procedure was carried out on one side of each rabbit (n = 20), and the contralateral muscle was the control. In the latissimus dorsi flap model, two perforators of the posterior intercostal vessels were ligated. In the biceps femoris flap model, a dominant vascular pedicle from the popliteal artery was ligated. After the 7-day delay period, the bilateral latissimus dorsi flaps (based on the thoracodorsal vessels) and the bilateral biceps femoris flaps (based on the sciatic vessels) were elevated. Animals were divided into three groups: part A, assessment of muscle flap viability at 7 days using the tetrazolium dye staining technique (n = 7); part B, assessment of vascular anatomy using lead oxide injection technique (n = 7); and part C, assessment of total and regional capillary blood flow using the radioactive microsphere technique (n = 6). The results in part A show that the average viable area of the latissimus dorsi flap was 96 +/- 0.4 percent (mean +/- SEM) in the delayed group and 84 +/- 0.7 percent (mean +/- SEM) in the control group (p < 0.05, n = 7), and the mean viable area of the biceps femoris flap was 95 +/- 2 percent in the delayed group and 78 +/- 5 percent in the control group (p < 0.05, n = 7). In part B, it was found that the line of necrosis in the latissimus dorsi flap usually appeared at the junction between the second and third vascular territory in the flap. Necrosis of the biceps femoris flap usually occurred in the third territory, and occasionally in both the second and the third territories. In Part C, total capillary blood flow in delayed flaps (both the latissimus dorsi and biceps femoris) was significantly higher than that in the control flaps (p < 0.05). Increased regional capillary blood flow was found in the middle and distal regions, compared with the control (p < 0.05, n = 6). In conclusion, ligation of either the dominant vascular pedicle in the biceps femoris muscle flap or the nondominant pedicle in the latissimus dorsi muscle flap in a delay procedure 1 week before flap elevation improves capillary blood flow and muscle viability. Vascular delay prevents distal flap necrosis in two rabbit muscle flap models.     

The effect of low-energy laser on skin-flap survival in the rat and porcine animal models.

Low-energy lasers are currently being used in the therapy of rheumatoid arthritis, chronic pain, muscle strain, and the promotion of wound healing in human and veterinary medicine. This study examined the effects of low-energy laser on skin-flap survival in a controlled interspecies study using the rat and porcine models. Twenty dorsal skin flaps based caudally were performed in 20 rats (10 laser-treated and 10 control flaps). The wounds were closed, and the flaps were sutured over the skin. Forty dorsal pig skin flaps based medially were raised in five pigs. The flaps were treated once per day for 10 days: 4 days preoperatively, the day of surgery, and 5 days postoperatively (30 s/cm3 per day). The average surviving rat flap surface area for the laser-treated flaps was 653 +/- 112 mm (mean +/- SD) and 580 +/- 60 mm in the control flaps, which was not significant (p greater than 0.05). In the porcine model, the average surviving area for the 20 laser-treated flaps was 949 +/- 174 mm, and the control average (n = 20) was 969 +/- 147 mm, also not significant. No beneficial effect was seen with low-energy laser preoperative and postoperative treatment of skin flaps in the rat and porcine models.     

Effect of hyperbaric oxygen and medicinal leeching on survival of axial skin flaps subjected to total venous occlusion

This study evaluates the effect of hyperbaric oxygen and medicinal leeching on axial skin flaps subjected to total venous occlusion. Axial epigastric skin flaps (3 x 6 cm) were elevated on their vascular pedicles in 40 male Wistar rats. Total venous occlusion was achieved by division of all veins draining the skin flap. Arterial inflow was left intact. Animals were randomly assigned to one of five groups: sham (n = 8); control, total venous occlusion only (n = 8); occlusion with hyperbaric oxygen (n = 8); occlusion with leeching (n = 8); occlusion with leeching and hyperbaric oxygen (n = 8). The hyperbaric oxygen protocol consisted of 90-minute treatments, twice daily, with 100% O2 at 2.5 atmospheres absolute for 4 days. The leeching protocol consisted of placing medicinal leeches on the congested flaps for 15 minutes, once daily, for 4 days. Laser Doppler measurements of flap perfusion were recorded preoperatively, postoperatively, and on postoperative days 1 and 3. The percentage of flap necrosis was evaluated on postoperative day 3. Mean percentage necrosis and mean laser Doppler readings were compared between both groups. The flaps in the sham group demonstrated 99 percent survival, whereas the flaps in the occlusion-only group demonstrated 100 percent necrosis. The flaps in the occlusion with oxygen, the occlusion with leeching, and the occlusion with oxygen and leeching groups demonstrated 1, 25, and 67 percent survival, respectively. Sham laser Doppler readings remained within normal limits. Laser Doppler readings in the occlusion-only and the occlusion with oxygen groups decreased to negligible levels on postoperative day 1, and on postoperative day 3 no perfusion was demonstrated. In both the occlusion with leeching and the occlusion with leeching and oxygen groups, there was also a significant decrease in laser Doppler measurements after surgery, but perfusion remained stable throughout the remainder of the study. This study demonstrates that hyperbaric oxygen alone is not an effective treatment for skin flaps compromised by total venous occlusion. The combination of leeching and hyperbaric oxygen treatment of total venous occlusion results in a significant increase in flap survival above that found with leeching alone. It appears that hyperbaric oxygen is effective because of the venous outflow provided by leeching as demonstrated by laser Doppler flow readings.     

Effect of vascular endothelial growth factor-induced angiogenesis on TRAM flap harvesting after abdominoplasty

In this study, the effect of intramuscular injection of human vascular endothelial growth factor (hVEGF) on neovascularization following abdominoplasty was investigated. Twenty-four Sprague-Dawley rats were divided into four groups (n = 6). Two control groups and two experimental groups were established. Abdominoplasty was performed in all rats, with division of all the perforator vessels. In the control groups, normal saline was injected into the rectus abdominis muscle, and in the experimental groups, 100 microg of VEGF and normal saline were injected into the rectus muscle. A transverse rectus abdominis musculocutaneous (TRAM) flap was harvested on day 20 and day 40 in both the control and experimental groups. The range of viability of the TRAM flap was, respectively, 0 to 20 percent (mean, 6.7 percent) and 0 to 25 percent (mean, 14.2 percent) in both short-term and long-term control groups (no VEGF injected). The study (VEGF) group demonstrated a viability of 50 to 80 percent (mean, 70 percent) for the short-term group and 50 to 85 percent (mean, 72.5 percent) in the long-term group. No wound infection was documented, and there were no deaths during the study period. There was no statistically significant difference between the short-term and long-term divisions of the groups (p < 0.01); however, significant differences were observed between the control and experimental groups (p < 0.01). The authors concluded that VEGF injection after abdominoplasty improved the percentage of TRAM flap viability. This method of delay/revascularization could be used for the difficult problem of flap viability following abdominoplasty and for high-risk patients.     

MSCT评价兔VX2乳腺种植瘤抗血管生成治疗的研究

探讨多层螺旋CT（multi—slice spiral computed tomography,MSCT）灌注成像与肿瘤血管内皮生长因子（vascular endothelial growth factor,VEGF）表达的相关性以评估兔VX2乳腺种植瘤抗血管生成治疗的疗效。将69R乳腺VX：瘤兔于肿瘤生长2周后随机分为对照组（生理盐水1、恩度组（Endostar）、cEF组[环磷酰胺（Cyclophosphamide C）、表阿霉素（EpirubicinE）和5-氟尿嘧啶（5．FluorouracilF）]、联合治疗CR（Endostar和CEF）。治疗2周后对瘤兔进行MSCT灌注扫描,获得血流量（bloodflow,BF）、血容量（bloodvolume,BV）、平均通过时间（meantransittime,MTT）及表面通透性（permeabilitysurface,PS）等灌注参数均值：随后取瘤组织进行免疫组化及Westernblot检测 VEGF蛋白表达情况。结果显示,对照组、CEF组、恩度组、联合治疗组BF、BV和Ps均与VEGF表达结果呈正相关（R对照组=0．896、0．680、0．765,RCEF组=0．877、0．876、0．852,R恩度组=0．804、0．924、0．888,R联合治疗组=0．780、0．735、0．744;P〈0．05）,MTT均与VEGF表达结果呈负相关（R对照组＝-0．591,RCEF组=0．678,R恩度组=0．793,R联合治疗组=-0．687;P〈0．05）。MSCT灌注参数与VEGF蛋白表达具有相关性,MSCT灌注参数可以反映肿瘤治疗后免疫组化与分子水平VEGF表达的变化,MSCT可以在体无创评价兔VX2乳腺种植瘤抗血管生成治疗的疗效。     

Increase in skin-flap survival by the vasoactive drug buflomedil

The effect of buflomedil to protect skin tissue from ischemia and necrosis was studied in random cutaneous flaps. Measurements were performed by intravital microscopy on the microcirculatory level of capillary perfusion in a flap model in the hairless mouse. In 30 hairless mice, single-pedicle flaps measuring 6 x 16 mm were raised perpendicular to the spine of the animal. This flap develops a reliable amount of necrosis at its distal edge over a period of 7 days. A group of 10 mice received intravenous injections of buflomedil in doses of 3 mg/kg per day diluted in 0.1 ml normal saline beginning 4 hours before flap elevation and for 6 consecutive days postoperatively. In addition, 10 further animals received the same treatment except that it was started 5 minutes after flap elevation. In 10 mice serving as controls, normal saline in equal volumes as in the experimental groups was applied. By means of intravital microscopy, functional vessel density (FVD) was determined in 2.5-mm increments from the flap's base to its distal edge at 1, 6, and 24 hours after elevation. Skin-flap survival was quantified by measuring the necrotic area on day 7 by means of digital planimetry. Functional vessel density was preserved in the distal flap of animals pretreated with buflomedil, revealing a higher functional vessel density at 10.0 mm (p less than 0.01), 12.5 mm (p less than 0.05), and 15.0 mm (p less than 0.001) from the flap's base as compared with controls.(ABSTRACT TRUNCATED AT 250 WORDS)