Adenovirus-mediated transforming growth factor-beta ameliorates ischemic necrosis of epigastric skin flaps in a rat model

BACKGROUND: Gene therapy has been recently introduced as a novel approach to treat ischemic tissues by using the angiogenic potential of certain growth factors. We investigated the effect of adenovirus-mediated gene therapy with transforming growth factor-beta (TGF-beta) delivered into the subdermal space to treat ischemically challenged epigastric skin flaps in a rat model. MATERIAL AND METHODS: A pilot study was conducted in a group of 5 animals pretreated with Ad-GFP and expression of green fluorescent protein in the skin flap sections was demonstrated under fluorescence microscopy at 2, 4, and 7 days after the treatment, indicating a successful transfection of the skin flaps following subdermal gene therapy. Next, 30 male Sprague Dawley rats were divided into 3 groups of 10 rats each. An epigastric skin flap model, based solely on the right inferior epigastric vessels, was used as the model in this study. Rats received subdermal injections of adenovirus encoding TGF-beta (Ad-TGF-beta) or green fluorescent protein (Ad-GFP) as treatment control. The third group (n = 10) received saline and served as a control group. 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. Just prior to flap elevation, the injections were given subdermally in the left upper corner of the flap. The flap was then sutured back to its bed. Flap viability was evaluated seven days after the initial operation. Digital images of the epigastric flaps were taken and areas of necrotic zones relative to total flap surface area were measured and expressed as percentages by using a software program. RESULTS: There was a significant increase in mean percent surviving area between the Ad-TGF-beta group and the two other control groups (P < 0.05). (Ad-TGF-beta: 90.3 +/- 4.0% versus Ad-GFP: 82.2 +/- 8.7% and saline group: 82.6 +/- 4.3%.) CONCLUSIONS: In this study, the authors were able to demonstrate that adenovirus-mediated gene therapy using TGF-beta ameliorated ischemic necrosis in an epigastric skin flap model, as confirmed by significant reduction in the necrotic zones of the flap. The results of this study raise the possibility of using adenovirus-mediated TGF-beta gene therapy to promote perfusion in random portion of skin flaps, especially in high-risk patients.     

Gene therapy with adenovirus-mediated VEGF enhances skin flap prefabrication

We investigated the feasibility in rats of enhancing skin-flap prefabrication with subdermal injections of adenovirus-encoding vascular endothelial growth factor (Ad-VEGF). The left saphenous vascular pedicle was used as a source for vascular induction. A peninsular abdominal flap (8 x 8 cm) was elevated as distally based, keeping the epigastric vessels intact on both sides. After the vascular pedicle was tacked underneath the abdominal flap, 34 rats were randomly divided into three groups according to treatment protocol. The implantation site around the pedicle was injected with Ad-VEGF in group I (n = 10), with adenovirus-encoding green fluorescent protein (Ad-GFP) in control group I (n = 14), and with saline in control group II (n = 10). All injections were given subdermally at four points around the implanted vessel by an individual blinded to the treatment protocol. The peninsular flap was sutured in its place, and 4 weeks later, an abdominal island flap based solely on the implanted vessels was elevated. The prefabricated island flap was sutured back, and flap viability was evaluated on day 7. Skin specimens were stained with hematoxylin and eosin for histological evaluation. In two rats from each group, microangiography was performed to visualize the vascularity of the prefabricated flaps. There was a significant increase in survival of prefabricated flaps in the Ad-VEGF group compared to the control groups: Ad-VEGF, 88.9 +/- 6.1% vs. Ad-GFP, 65.6 +/- 9.4% (P < 0.05) and saline, 56.0 +/- 3.4% (P < 0.05). Sections from four prefabricated flaps treated with Ad-GFP revealed multiple sites of shiny deposits of green fluorescent protein around the area of local administration 1 day and 3 weeks after gene therapy. Histological examination done under high-power magnification (x400) with a light microscope revealed increased vascularity and mild inflammation surrounding the implanted vessel in all groups. However, we were unable to demonstrate any significant quantitative difference with respect to vascularity and inflammatory infiltrates in prefabricated flaps treated with Ad-VEGF compared with controls. Microangiographic studies showed increased vascularity around the implanted pedicle, which was similar in all groups. However, vascularization was distributed in a larger area in the prefabricated flaps treated with Ad-VEGF. In this study, the authors demonstrated that adenovirus-mediated VEGF gene therapy increased the survival of prefabricated flaps, suggesting that it may allow prefabrication of larger flaps and have the potential to reduce the time required for flap maturation.     

Comparison of the effectiveness of gene therapy with vascular endothelial growth factor or shock wave therapy to reduce ischaemic necrosis in an epigastric skin flap model in rats.

The effect of gene therapy with adenovirus-mediated (Ad) vascular endothelial growth factor (VEGF) was compared to that of shock wave (SW) therapy on skin flap survival in a rat model, using the epigastric skin flap, based solely on the right inferior epigastric vessels. Thirty male Sprague-Dawley rats were randomly divided into three groups (SW-group, Ad-VEGF-group, and Control-group) of 10 rats each. Immediately after surgery, the SW-group was administered 2500 impulses at 0.15mJ/mm(2), in the Ad-VEGF-group injections were made to the subdermal space whereas the Control-group received no treatment. Flap viability was evaluated on day 7 after the operation. Standardised digital pictures of the flaps were taken and transferred to the computer, and necrotic zones relative to total flap surface area were measured and expressed as percentages. Overall, significantly smaller areas of necrotic zones were noted in the SW-group and the Ad-VEGF-group compared with the Control-group (SW-group: median 2.23% (range: 0-5.1) versus Control-group: median 17.4% (range: 11.8-22.8) (p<0.05); Ad-VEGF-group: median 9.25% (range: 7.6-11.9) versus Control-group: median 17.4% (range: 11.8-22.8) (p<0.05)). Furthermore, in the SW-group, areas of necrotic zones were significantly smaller than in Ad-VEGF-group (SW-group: median 2.23% (range: 0-5.1) versus Ad-VEGF-group: median 9.25% (range: 7.6-11.9) (p<0.05)). We conclude that treatment with SW enhances epigastric skin flap survival significantly more than Ad-VEGF treatment and also represents a feasible and cost effective technique to improve blood supply in ischaemic tissue.     

Adenovirus-mediated angiopoietin-1 gene therapy enhances skin flap survival

This experimental study investigates the feasibility of pretreating the abdominal skin of a rat with subdermal injections of adenovirus encoding angiopoietin-1 in order to improve postoperative survival of the skin flap. An epigastric skin flap was used as the model in this study. Rats received subdermal injections of adenovirus encoding either angiopoietin-1 (treatment group) or green fluorescent protein (treatment control), or they received no treatment (control group). Subdermal injections were made 2 days prior to surgery, and skin flap survival was assessed 7 days afterwards as a percentage of necrotic area over total skin flap area. The treatment group which received adenovirus-mediated angiopoietin-1 had a median percent necrotic area of 11.01%, a significant decrease from the control group, which had a median percent necrotic area of 32.24% (P < 0.001). The results of this study suggest the possibility of using adenovirus-mediated angiopoietin-1 gene therapy to promote therapeutic angiogenesis in patients who undergo reconstructive procedures.     

Effect of adenovirus-mediated gene transfection of vascular endothelial growth factor on survival of random flaps in rats

Objective: To evaluate the effect of local application of vascular endothelial growth factor ( VEGF ) via adenovirus-mediated gene transfer on survival of full thickness flaps selected randomly in rats.Methods: Thirty Sprague-Dawley rats weighing 480-520 g were used in this study. A dorsal flap (8 cm × 2 cm) in full thickness with the pedicle located at the level of the iliac crest was designed. Then the rats received 1 012 pfu replication-deficient recombinant adenovirus carrying VEGF ( AdCMV-VEGF group, n = 10 ), 1012 pfu recombinant β-galactosidase adenovirus ( AdCMV-Gal group, n = 10) and 1 ml saline (saline group, n = 10), respectively, in the distal two thirds of the proposed flap by means of subdermal injection at 8 different locations. Three days after treatment, the flaps were elevated as originally designed and sutured back in situ. The survival rate of the flaps was evaluated on day 7 after operation.Results: The survival rate of the flaps in the AdCMV-VEGF group increased significantly as     

Comparison of the effectiveness of gene therapy with transforming growth factor-β or extracorporal shock wave therapy to reduce ischemic necrosis in an epigastric skin flap model in rats

The induction of neoangiogenesis by exogenous growth factors in failing skin flaps has recently yielded promising results. Gene transfer with virus vectors has been introduced as a highly capable route of administration for growth factors, such as vascular endothelial growth factor or fibroblast growth factor. Extracorporal shock waves (ESW) deliver energy by means of high amplitudes of sound to the target tissue and have been shown to induce angiogenesis. We compared the effectiveness of gene therapy with adenovirus-mediated transforming growth factor-beta (TGF-beta) and ESW therapy to treat ischemically challenged epigastric skin flaps in a rat model. Thirty male Sprague-Dawley rats were divided into three groups of 10 each with an 8 x 8 cm epigastric skin flap. Rats received either subdermal injections of adenovirus (Ad) encoding TGF-beta (10(8) pfu) or ESW treatment with 750 impulses at 0.15 mJ/mm2. The third group received no treatment and served as a control group. Flap viability was evaluated after 7 days and digital images of the epigastric flaps were taken and areas of necrotic zones relative to total flap surface area calculated. Histologic evaluation and increased angiogenesis were confirmed by CD31 immunohistochemistry. Overall, there was a significant increase in mean percent surviving area in the Ad-TGF-beta group and the ESW group compared to the control group (ESW group: 97.7 +/- 1.8% vs. Ad-TGF-beta: 90.3 +/- 4.0% and control group: 82.6 +/- 4.3%; p < 0.05). Furthermore, in the ESW group mean percent surviving areas were significantly larger than in the Ad-TGF-beta group (ESW group: 97.7 +/- 1.8% vs. Ad-TGF-beta: 90.3 +/- 4.0%; p < 0.05). Flap vascularization was increased by Ad-TGF-beta and ESW with numerous vessels, however, there was no significant difference between the two treatment groups. We conclude that treatment with ESW enhances epigastric skin flap survival significantly more than Ad-TGF-beta treatment and thus represents a modality that is feasible, cost-effective, and less invasive compared to gene therapy with growth factors to improve blood supply to ischemic tissue.     

VEGF gene therapy for the survival of transplanted fat tissue in nude mice.

The objective of this study was to determine the effects of adenovirus-mediated vascular endothelial growth factor (Ad-VEGF) on the angiogenesis and survival of free-fat tissue transplantation in nude mice. Thirty 6-week-old CD-1 nude male mice were injected with 1ml fat tissue (harvested by suction-assisted lipectomy from the breast of humans) in the subcutaneous of scalp and were randomised into three groups of 10 animals each. Group 1 was the study group, in which Ad-VEGF was mixed with transplanted fat tissue and injected into mice. In group 2, adenovirus-mediated green fluorescent protein (Ad-GFP) gene was mixed with transplanted fat tissue and injected into the mice. In group 3, normal saline alone was used. Both group 2 and group 3 are control groups. The animals were euthanised 15 weeks after the procedure. The fat survival weight and volume of the study group were significantly greater than those of two control groups (p<0.05). Light microscopical examination of haematoxylin and eosin-stained slides of the dissected fat 15 weeks after injection was performed in group 1 and group 2. Less cyst formation and fibrosis, indicating improved quality of the injected fat, can be obtained by the addition of Ad-VEGF. Vascular density was evaluated at the microvascular level through the use of light microscopic sections of the central part of the fat tissue at 15 weeks after injection by von Willebrand factor staining. Histological evaluation showed that capillary density increased markedly in the study group mice. Mice of the study group disclosed significantly higher VEGF protein levels detected by ELISA assay of plasma samples obtained from the mice after the fat injection (day 1, 4, 7 and 28; p<0.01) at each time point than the mice of the two control groups. The findings reported in this study indicate that the VEGF gene therapy can enhance the survival and the quality of grafted fat tissue, which may be due to induction of angiogenesis.     

Enhancement of ischemic flap survival by prefabrication with transfer of exogenous PDGF gene

Treatment of skin flaps by means of gene therapy has been introduced recently as a novel approach to enhance viability of ischemic skin flaps. Transfer of the platelet-derived growth factor (PDGF) to enhance survival of the ischemic skin flap has not been explored. In this study, the authors investigated the effect of the transfer of the PDGF cDNA on survival and vascularity of the ischemic random flap in a rat model, and compared the effects of PDGF gene therapy to those of vascular endothelial growth factor (VEGF) gene therapy. A total of 45 adult Sprague-Dawley rats were randomly divided into four groups. The PDGF gene therapy group (n=10) received the plasmid containing the PDGF cDNA with liposome injected to the dermis of the flap. A saline control group (n=10) received physiologic saline only, and the vector control group (n=10) received liposome plus vector without the PDGF gene segment. In the fourth group (n=15), the VEGF gene was transferred to the flap. Seven days later, a dorsal random flap including the injection area was raised. One rat each from the saline and vector control groups died during the study period and were excluded. The viability of the flap and vascularity within the flaps were assessed 7 days after flap elevation. The PDGF plasmid-treated flaps had significantly greater survival area (60.8+/-7.8 percent) compared with the flaps treated with saline (52.3+/-5.0 percent) and those treated with liposome and vector (50.7+/-5.9 percent). PDGF gene therapy had effects on survival of the flap similar to VEGF gene therapy (57.6+/-5.2 percent, after transfer of VEGF cDNA). Neovascularization with the flap tissues was confirmed by immunohistochemical staining of von Willebrand factor, a marker specific for angiogenesis. The number of newly-formed blood vessels in the transgenic flaps was significantly greater than that of the vessels in the flaps receiving the saline. The findings of this study indicate that transfer of the PDGF cDNA effectively enhances neovascularization of the ischemic skin flap and increases the viability of the flap, and transfer of the PDGF gene is as efficient as transfer of the VEGF gene in improving viability of the skin flap. This study suggests that PDGF gene therapy may be a novel strategy for the treatment of ischemic skin flaps.     

腺病毒-VEGF基因重组体促进预构皮瓣成活的实验研究

目的：应用大鼠预构皮瓣模型,探讨基因治疗技术产生的血管内皮生长因子促进预构皮瓣血管新生和皮瓣存活的可能性,为临床上寻找加速预构皮瓣成熟的新方法提供实验依据。方法：20只SD大鼠每只腹部两侧各构建一个预构皮瓣,共构建40个皮瓣,每只大鼠两侧皮瓣按随机原则进行不同的处理,分别归于实验组或对照组,每组各20个皮瓣。于大鼠腹部两侧各标记3cm×2cm矩形预构区,短边平行于腹股沟韧带,自尾侧短边中点向后纵向切开后肢皮肤,剥离出长约2cm的股动静血管束,远端结扎切断。在两侧预构区域的中轴线上,于真皮与肉膜层间各制作一皮下隧道,实验组的隧道壁皮下组织内注射携带有VEGF基因的腺病毒,同法向所有对照组的隧道壁软组织内注射等量生理盐水。将已剥离好的血管束向颅侧翻转置入相应皮下隧道内。所有已植入股血管的预购区域2周后均被制成以植入血管束为蒂的岛状皮瓣,从两组中各取一个皮瓣进行免疫组化染色,观察有无VEGF生成,其余岛状皮瓣均缝回原处。形成岛状皮瓣后第七天观察皮瓣存活及血管新生情况。结果：实验组与对照组的皮瓣平均存活率分别为（90．48±1．89）％、（69．75±2．36）％,其差异有统计学意义（P〈0．01）;血管放射显影图上,实验组植入血管周围见广泛白色显影,尤以血管两端明显,而对照组新生血管显影仅局限于植入血管周围;组织学切片显示实验组植入血管周围新生血管丰富,以毛细血管为主,并见肉芽成份,对照组新生血管相对较少,两组间新生小血管管腔大小则无明显差异;免疫组化检测显示仅实验组皮瓣中有VEGF表达。结论：腺病毒-VEGF基因重组体能通过促进预构皮瓣的血管新生,增加预构皮瓣的存活率。     

重组腺病毒介导的血管内皮生长因子165基因增强大鼠横形腹直肌肌皮瓣活力的研究

目的探讨重组腺病毒介导的血管内皮生长因子165(adenovirusmediatedvascularendothelialgrowthfactor165,Ad-VEGF165)基因,增强大鼠横形腹直肌肌皮瓣(transverserectusabdominismusculocutaneousflap,TRAM)局部缺血区域活力的基因治疗。方法SD大鼠30只,制备30mm×80mm以下腹部血管作为血管蒂的右侧矩形TRAM皮瓣,随机平均分成5组,分别在大鼠TRAM皮瓣皮下、皮下+腹直肌及腹直肌局部注射Ad-VEGF165(1～3组:治疗组),同时在TRAM皮瓣皮下+腹直肌局部注射重组腺病毒载体介导的绿色荧光蛋白(adenovirusmediatedgreenfluorescentprotein,Ad-GFP,4组:重组腺病毒自身对照组)和DMEM培养液(5组:空白对照组),于注射后第7天手术,术后第7天检测各组大鼠TRAM皮瓣成活面积、CD34微血管密度(microvasculardensity,MVD)、VEGF免疫组织化学染色和原位杂交组织化学(insituhybridizationhistochemistry,ISHH)。结果11～3组TRAM皮瓣成活面积分别为14.19±2.77、15.18±2.18、8.30±1.28cm2,均较4、5组4.12±1.86、3.60±1.95cm2明显增加,且差异有统计学意义(P<0.05);21～3组TRAM皮瓣CD34MVD均高于4、5组,差异有统计学意义(P<0.05);31～3组VEGF免疫组织化学染色和ISHH呈阳性表达。结论重组腺病毒载体介导的VEGF165基因局部注射,能增加大鼠TRAM皮瓣局部缺血区域成活面积。     

Enhancement of muscle flap hemodynamics by angiopoietin-1

Angiopoietin-1 (Ang-1) constitutes a novel family of endothelial cell-specific angiogenic factors. Ang-1 functions mainly in remodeling, maturation, and stabilization of blood vessels. Its direct role in the process of angiogenesis remains unknown. The authors designed an experimental study to investigate the angiogenic potential of Ang-1 and to determine its hemodynamic effects on the cremaster muscle flap model in the rat. Adenovirus-mediated gene therapy was used for delivery of Ang-1. The study sample included 45 male Sprague-Dawley rats weighing 200 to 250 g. After the cremaster muscle tube flaps were prepared, rats were randomized into three different groups of 15 animals. In group I (the control), the flaps received phosphate-buffered saline (PBS). In group II, flaps were treated with adenovirus vector encoding Ang-1 (Ad-Ang-1). In group III, flaps received a control gene encoding green fluorescein protein (Ad-GFP). All treatments were administered via intra-arterial injections of either viral particles (10(8) placque-forming units) or PBS. The external iliac artery was used for this purpose. The cremaster tube flap was then preserved in a subcutaneous pocket in the lower limb. The tube flap was withdrawn from the limb on days 3, 7, and 14 after intra-arterial injection to evaluate microcirculatory measurements such as red blood cell velocity, vessel diameter, capillary density, and microvascular permeability by intravital microscopy. Evaluations were performed by an investigator who was blinded to treatment groups. In a series of control experiments performed with Ad-GFP, adenoviral gene expression was evidenced by the observation of shiny GFP deposits along the vessel walls under fluorescence microscopy throughout the whole cremaster flap 2 days after transfection. At day 3 there was no evidence of any differences in capillary density and permeability index (PI). At day 7, the functional capillary density was significantly higher in the Ad-Ang-1-treated group compared with the control and the Ad-GFP groups (10/hpf +/- 2 vs. 7/hpf +/- 0.5, p = 0.006; 5/hpf +/- 1.6, p = 0.0001). The PI in the Ad-Ang-1-treated group was significantly lower compared with the Ad-GFP-treated group (1.1/hpf +/- 0.1% vs. 1.4/hpf +/- 0.1%, p = 0.0005). At 14 days, the number of the flowing capillaries was significantly higher in the Ad-Ang-1-treated group compared with the control and the Ad-GFP-treated groups (13/hpf +/- 1.7 vs. 9/hpf +/- 2 and 6/hpf +/- 1.3, p = 0.0001). The microvascular PI was significantly lower in the Ad-Ang-1-treated group compared with the Ad-GFP-treated group (1.3/hpf +/- 0.2% vs. 1.8/hpf +/- 0.5%, p = 0.004). Histologically, the cremaster flaps revealed focal and mild inflammation regardless of the treatment and time point of evaluation. There was evidence of vasculitis in muscles pretreated with Ad-GFP and Ad-Ang-1. In summary, in the Ad-Ang-1-treated cremaster flaps, functional capillary density increased from 46% at day 7 to 98% at day 14 when compared with the control group (p < 0.0001). In conclusion, in this experimental muscle flap model, Ad-Ang-1 treatment proved to be a successful method of angiogenic therapy, providing a long-lasting angiogenic effect over a period of 14 days. The increased capillary perfusion accompanied by the formation of more stable and mature vessels resistant to fluorescein isothiocyanate-conjugated albumin leakage may serve as in vivo evidence that Ang-1 therapy improves skeletal muscle flap hemodynamics. These exciting findings raise the possibility that Ang-1 may have implications for therapeutic angiogenesis. To the authors' knowledge, their study demonstrates for the first time the feasibility of intravascular gene therapy using a virus vector in an attempt to enhance muscle flap hemodynamics.     

脂质体介导血管内皮生长因子对不同时间断蒂大鼠随意型皮瓣的影响

目的 通过血管内皮生长因子基因对大鼠随意型皮瓣的转染,探讨基因治疗对不同时间断蒂的大鼠随意型皮瓣成活的影响.方法 以SD大鼠为实验模型制作背部随意型皮瓣,实验组注入脂质体包裹的PcDNAVEGF165(目的 基因组),对照组分别注入PcDNA(空白质粒组)和生理盐水(生理盐水组),于用药后1、3、5、7 d,每组每时相点分别随机选取10只断蒂,断蒂后7 d处死大鼠,观察下述指标:①皮瓣成活率.②皮瓣组织标本行常规HE染色检测平均微血管数目及内径.③行VEGF免疫组织化学染色检测VEGF表达情况.④取皮瓣组织标本在电镜下观察超微结构.结果 ①皮瓣成活率:1、3、5、7 d断蒂实验组皮瓣成活率分别为(45.45±12.24)%、(82.95±3.81)%、(85.00±3.38)%、(85.96±3.25)%.1 d断蒂实验组与对照组比较差异无统计学意义(P>0.05),3、5、7 d断蒂各实验组明显高于相应对照组(P<0.05),3、5、7 d断蒂各实验组则随着断蒂时间的延迟差异无统计学意义(P>0.05).②平均微血管数目及内径:各实验组与相应对照组比较差异有统计学意义(P<0.05).③各实验组VEGF染色深度明显高于对照组(P<0.05).④超微结构:实验组内有新生血管形成,内皮细胞内可见较多粗面内质网,线粒体等结构,组织内成纤维细胞增多,细胞合成代谢旺盛.结论 皮下注射脂质体介导VEGF基因可提高皮瓣成活率,促进早期断蒂,是一种简单,高效,经济,相对安全的基因治疗方法.     

血管内皮生长因子促进跨区供血反流轴型皮瓣成活的实验研究

目的研究血管内皮生长因子（vascular endothelial growth factor，VEGF）的局部皮下注射对大鼠背部跨区供血反流轴型皮瓣成活的影响及效果。方法取20只SD大鼠，制备8cm×2cm大鼠背部跨区供血反流轴型皮瓣模型，随机分成两组，每组10只。实验组：于皮瓣远端7．5cm及6．5cm处共选择4个对称位点，分别予100ng／100μlVEGF溶液50μl；对照组：每一位点予生理盐水50μl。术后1～7d行皮瓣大体观察，并于7d处死大鼠，切取皮瓣，行皮瓣成活率测定、组织学观察及血管密度检测。结果大体观察，实验组皮瓣成活面积明显大于对照组，实验组皮瓣成活面积15．55±0．27cm^2，对照组13．42±0．57cm^2，差异有统计学意义（P〈0.01）。组织学观察，实验组皮瓣血管密度34．40±3．75个／10倍光镜下视野，对照组21．00±3．16个／10倍光镜下视野，差异有统计学意义（P〈0.01）。镜下见实验组有大量新生肉芽组织形成，胶原纤维排列规则，成纤维细胞较多，炎性细胞浸润程度轻；对照组新生肉芽组织少，胶原纤维凝集成块，成纤维细胞少，炎性细胞浸润程度重。结论VEGF在皮瓣成活早期，通过促进缺血皮瓣新生血管形成，增加血管数量，改善缺血组织的血液供应，促进皮瓣成活；在皮瓣形成时局部、单次、足量应用VEGF是促进跨区供血反流轴型皮瓣远端成活的有效方法。     

腺病毒载体介导人VEGF cDNA促进随意型皮瓣成活的实验研究

目的探讨以腺病毒载体局部应用血管内皮细胞生长因子基因对大鼠缺血随意型皮瓣成活的影响。方法30只SD大鼠随机分成3组,每组10只,于鼠背设计蒂在尾侧髂嵴水平的随意型皮瓣,面积为2cm×8cm,A组皮瓣远端23顺皮瓣长轴分10个点真皮下注入1012PFU的携带人血管内皮细胞生长因子基因的腺病毒(AdCMVVEGF),B组同法注入同剂量的携带β半乳糖苷酶基因的腺病毒(AdCMVGal),C组注入1ml的生理盐水,3d后掀起皮瓣并原位缝合,术后7d观察实验结果。结果每组皮瓣成活面积的百分比分别为A组(8591±254)%,B组为(5956±118)%,C组为(6148±109)%,A组成活率显著高于B组和C组,差异有非常显著性意义(P<001),B组与C组比较差异无显著性意义(P>005)。原位杂交、免疫组化显示,应用AdCMVVEGF治疗的成活皮瓣中,VEGF有表达,组织学切片显示,AdCMVVEGF治疗皮瓣中肉芽组织增生,新生血管大量形成。结论以腺病毒为载体局部应用VEGF基因能增加缺血皮瓣的成活面积,该法具有高效的特点。     

皮下注射血管内皮生长因子基因提高大鼠背部随意皮瓣活力的实验研究

目的 探索基因治疗在组织移植方面的方法。应用 构建并体外扩增表达质粒pcDNA3.1/Zeo( )-VEGF165，以之直接皮下注射转染SD大鼠背部随意皮瓣模型。通过与对照组相比较，观察其对皮瓣活力的影响，并采集组织行免疫组化检查。结果 基因转染组在毛细血管周围及肌间隙可见VEGF的沉积，与对照组相比皮瓣的平均成活面积显著增加。结论 皮下注射的基因在组织中能够表达VEGF，增加皮瓣活力，是一种简单有效的基因治疗方法。     

Reliability of island flaps raised after superficial and deep burn injury

In select cases, to prevent any functional loss and to initiate early function during the early burn period, the reconstructive procedure of choice may be flap coverage. In these circumstances, when the ideal flap donor site is burned, the clinician may be hesitant to raise this flap because of questionable flap survival. The authors conducted this study to determine whether a superficially or deeply burned skin island flap would survive when elevated during the early postburn period. If these flaps are usable, they could expand the options available for burn wound coverage. They used a rat epigastric island flap model, and divided 50 study animals into two groups. In group 1 (N = 25), the right epigastric flap site was burned superficially and the left side was left uninjured. Island flaps were raised on both sides 4 days after the burn injury. The flaps were then sutured back into their original sites, and were evaluated 5 days after the surgery. In group 2 (N = 25), the right epigastric flap site was burned deeply and the left side was left uninjured. Island flaps were raised 4 days after the burn injury on both sides, as in group 1. The flaps were then sutured back into place and were evaluated 5 days after the surgery. All of the control flaps on the rats' left sides survived in both groups. In addition, all the superficially burned flaps survived in group 1 (100%), and 21 of the deeply burned flaps survived in group 2 (84%). There was no significant difference between superficially and deeply burned flaps with regard to survival, and the burned flaps were as successful as the unburned control flaps in both groups (p = 0.11). Skin island flaps elevated after superficial or deep burn injury are reliable in this animal model.     

The effect of single administration of vascular endothelial growth factor or L-arginine on necrosis and vasculature of the epigastric flap in the rat model.

OBJECTIVES: Vascular endothelial growth factor (VEGF) and nitric oxide (NO) produce vasodilation, induce angiogenesis, and improve survival of surgical flaps. We used the rat epigastric skin flap to study the effect of a single intra-arterial dose of VEGF or L-arginine, a substrate for NO production, on flap regional necrosis and pedicle dependence of flap perfusion. METHODS: In 30 Sprague-Dawley rats an 8 x 8 cm2 skin flap, consisting of four vertical zones marked A through D (right to left), based on the proximal right inferior epigastric vessels was raised. Subsequently, 1 ml of either saline (control, n =10), 5 microg VEGF (VEGF, n = 10), or 50 mg of L-arginine (L-arginine, n = 10) was injected into the arterial pedicle by cannulating the right saphenous artery, and the flap was resutured in place. After 8 days, the animals were perfused systemically with 15 microm coloured fluorescent microspheres before (blue) and after (yellow-green) ligation of the right inferior epigastric vascular pedicle. After sacrifice, the area of flap necrosis was measured in each zone by templates and weight-to-surface ratio, and the flap zones were harvested and processed for determination of fluorescence and blood flow. RESULTS: Administration of VEGF or L-arginine resulted in decreased total and regional (zone D) flap necrosis (ANOVA <0.001). The total and regional flap shrinkage was greater in the experimental groups (ANOVA <0.02). While VEGF and L-arginine decreased the percentage of necrosis in the zone most distal to the pedicle (ANOVA <0.01) only L-arginine diminished percentage of total flap necrosis (p = 0.04). In the VEGF group, total and regional flap perfusion did not change after pedicle ligation, but perfusion decreased significantly in zones B through D in the L-arginine treated rats. CONCLUSION: Single intra-pedicle administration of VEGF or L-arginine decreased necrosis of the epigastric skin flap at 8 days postoperatively, but flap shrinkage also increased in the zone with the greatest degree of necrosis. Perfusion data suggest that beneficial effects of VEGF and L-arginine on flap survival may be based on different mechanisms.     

髂腹股沟带蒂真皮下血管网皮瓣修复手部皮肤脱套伤

目的应用髂腹股沟带蒂真皮下血管网皮瓣修复手部皮肤脱套伤.方法将带旋髂浅血管或腹壁浅血管为蒂的髂腹股沟皮瓣的远端1/3～1/2修剪成带真皮下血管网薄皮瓣,修复除拇指以外的手部大面积皮肤缺损.结果临床应用9例,皮瓣全部成活,外形及运动功能恢复满意.结论带蒂髂腹股沟真皮下血管网皮瓣修复手部皮肤脱套伤是简单、有效的方法.     

Evaluation of the Effect of Sildenafil and Vascular Endothelium Growth Factor Combination Treatment on Skin Flap Survival in Rats

BACKGROUND: Distal ischemic necrosis of surgical flaps remains a challenging problem for the reconstructive surgeon. Recent studies have shown that either sildenafil or vascular endothelium growth factor (VEGF) treatment significantly improves ischemic skin flap viability. In this study, the effect of the combination of sildenafil and VEGF165 was evaluated on a rat skin flap model using orthogonal polarization spectral imaging and histologic analysis. METHODS: Rats were assigned to either a sham (n = 31), vehicle (n = 24), sildenafil (n = 24), VEGF (n = 23), or sildenafil and VEGF combination treatment (n = 21) groups. Distances from the distal end of the flap to avascular, stasis, and normal capillary blood flow zones were determined using orthogonal polarization spectral imaging on a skin flap model. Vessel density assessment was done at 7 days post surgery. RESULTS: Imaging analysis showed significant reduction in avascular and stasis areas in sildenafil and VEGF combination-treated groups at 7 days post surgery (p < 0.05). The combination-treated group, however, was not significantly different when compared to the group treated with sildenafil only. The sildenafil-treated group showed a significant (p < 0.05) reduction in both areas at day 7 compared to the VEGF and control groups. Histologic analysis showed no significant differences in vessel density between the groups. CONCLUSION: The combination of sildenafil and VEGF decreases the extent of avascular and stasis zones in skin flaps. The skin flap improvement seen with the combination treatment was similar to the sildenafil treatment alone suggesting that enhanced flap survival was due solely to the effect of sildenafil.     

The effect of trimetazidine on the survival of rat island skin flaps subjected to ischemia-reperfusion injury

The effect of trimetazidine (TMZ) on flap ischemia-reperfusion injury was investigated in rat inferior epigastric artery flaps. Twenty-six rats, divided into four experimental groups-nonischemic group (group 1, N = 5), ischemic control group (group 2, N = 7), preischemic TMZ-treated group (group 3, N = 7), and postischemic TMZ-treated group (group 4, N = 7)-were used. Rat inferior epigastric artery flaps were rendered ischemic by occluding the feeding femoral artery, and they were reperfused by releasing the clamps after 11 hours in groups 2 through 4. Group 3 rats were given TMZ (3 mg per kilogram, intravenously) diluted in saline before application of the clamp, and group 4 rats were given TMZ before clamp removal. Flap survival was scored on postoperative day 8. All flaps in the nonischemic control group (group 1) survived completely. The ischemic control group (group 2) demonstrated a 6.3 +/- 4.3% survival area. In the preischemic TMZ group (group 3) the mean survival area was 76.9 +/- 6.1%, and in the postischemic TMZ group (group 4) it was 76.8 +/- 5.6%. TMZ-treated flaps showed a significant increase in survival area regardless of the time of administration (p = 0.001, group 3 vs. group 2; p = 0.001, group 4 vs. group 2). This finding suggests that TMZ has a beneficial effect on the prevention or treatment of arterial ischemic flaps.