Remote ischemic preconditioning improves the survival of rat random-pattern skin flaps

Random-pattern flap transfer is one of the most popular procedures for covering soft tissue defects. Ischemic preconditioning is a protective endogenous mechanism capable of reducing ischemia-reperfusion injury, and it has been shown that preconditioning by proximal pedicle clamping can improve flap survival. However, the method is not suitable for random-pattern flap transfer in the clinical setting. The present study evaluates the effect of ischemic preconditioning of Wistar rat hind limbs upon dorsal random-pattern skin flap survival. Ischemic preconditioning was induced by ischemia of the right hind limb during 10 min, followed by 30 min of reperfusion. Thirty-two animals were divided in two groups. In group 1, a dorsal random-pattern skin flap measuring 2 × 7 cm was raised immediately after the induction of ischemic preconditioning. The animals in group 2 (controls) received the same treatment, but without ischemic preconditioning. The survival area was defined as the surface of the viable tissue (square centimeter) on the fifth postoperative day. The average survival area was 6.57 ± 0.18 cm² in group 1 and 4.44 ± 0.21 cm² in group 2. All preconditioned animals presented significantly higher flap survival areas than the controls (p ≤ 0.01, Student’s t test). Our findings show that flap necrosis was reduced by the induction of ischemic preconditioning in a body area distant from the flap harvest site, and that ischemic preconditioning has a systemic protective effect on dorsal random-pattern skin flaps and increase survival. A better understanding of the mechanism involved may improve the clinical condition of patients requiring random-pattern skin flap transfer, especially in high-risk groups.     

Vascular endothelial growth factor gene therapy in ischaemic rat skin flaps.

Gene therapy with the complementary DNA (cDNA) of the angiogenic cytokine vascular endothelial growth factor (VEGF) has emerged as a promising strategy in the treatment of myocardial and lower-limb ischaemia. The objective of this study was to determine whether these principles could be applied to a recognised model of skin-flap ischaemia. Plasmid vectors including the cDNA of green fluorescent protein (GFP) and one of three VEGF isoforms (A165, B167 or B186) were constructed, and their base sequences confirmed. GFP expression was used as a marker of successful in vitro transfection of human endothelial cells with each plasmid. The plasmids were then administered subcutaneously to rat abdominal skin flaps surgically rendered ischaemic, and the percentage of viable tissue was assessed at 1 week. Angiograms of the flaps and histological preparations of flap tissue were assessed for evidence of angiogenesis. The survival of flaps treated with VEGF A165 or B167 cDNA was significantly greater than that of controls (P < 0.05). The survival of flaps treated with VEGF B186 cDNA was greater than that of controls, but statistical significance was not reached. Angiograms and microvessel density counts failed to produce evidence of angiogenesis. With improved delivery strategies, VEGF may have a role in the management of surgical ischaemia.     

Improvement of skin paddle survival by application of vascular endothelial growth factor in a rat TRAM flap model

The effect of vascular endothelial growth factor (VEGF) on skin flap survival and its ability to induce a pharmacological delay by promoting angiogenesis in a flap was studied in a rat transverse rectus abdominis musculocutaneous flap, using a 3 x 8-cm skin paddle with the inferior epigastric vessels as its main vascular supply. Forty-three Sprague-Dawley rats were divided into four groups. In group 1, VEGF was injected into the femoral vein after the flap was elevated. In group 2, VEGF was injected intra-arterially into the flap through the superior epigastric artery after the flap was elevated. In group 3, VEGF was injected into the subcutaneous fascial layer in the area where the flap would be dissected, and the flap was then raised 7 days after injection. In group 4, the flap was dissected and replaced, using saline injection as the control. On postoperative day 5, the survival area of each skin paddle was measured and the flap was harvested for histological analysis. The results showed that the mean survival area +/- standard deviation for the skin paddle was 6.82 +/- 4.89 cm2 (28.4 +/- 20.4% of the whole skin paddle) in the control group, and 4.2 +/- 3.0 cm2 (17.5 +/- 12.5%) and 6.02 +/- 5.97 cm2 (25.1 +/- 24.9%) in the groups with VEGF systemic and intra-arterial administration respectively. The skin survival area in the group with preoperative subcutaneous administration of VEGF was 17.85 +/- 2.88 cm2 (74.4 +/- 12%), which was significantly higher than the other three groups (p < 0.01). Histological semiquantitative analysis showed increased neovascularization in the flap treated with VEGF preoperatively. The data demonstrate that preoperative treatment with VEGF can induce angiogenesis and enhance skin paddle survival in a musculocutaneous flap.     

Salutary effects of radiopaque contrast media on the survival of random-pattern skin flaps in the rat: an experimental study

The radiopaque contrast medium diatrizoate, has a vasodilator effect so that it is used in sudden-deafness secondary ischemic injury. However, ischemic problems are encountered, especially when longer flaps are elevated. A longer flap also has ischemic and relatively ischemic tissue, and may obtain some benefit from contrast media. Forty male Sprague-Dawley rats, weighing about 350-400 g, were used, and randomly divided into four groups (n = 10 rats each group): group 1 was the control, group 2 the diatrizoate, group 3 the iopamidol, and group 4 the iothalamate group. A rectangular 3 x 10 cm caudally based dorsal skin flap was elevated, and sutured back to its original place. In the control group, no pharmacologic agent was administered. Sodium-meglumine-diatrizoate 10 mg/kg/day was administered parenterally in the first experimental group (diatrizoate group); iopamidol 10 mg/kg/day in the second experimental group (iopamidol group); and iothalamate sodium 10 mg/kg/day in the third experimental group (iothalamate group) for 7 postoperative days. On postoperative day 7, all flaps were photographed, and the area of flap survival was measured by using a polar planimeter. The results were statistically evaluated with the Kruskal-Wallis test and Mann-Whitney U-test (P = 0.05). The mean flap survival ranged from 79% in the iopamidol group to 83% in the diatrizoate group, and was significantly greater in all experimental groups (P < 0.05) compared to the control group (59%). There was no significant difference between experimental groups (P < 0.05). We believe that radiopaque contrast media have a beneficial effect in improving skin flap viability when distal flap necrosis is a potential complication of longer flaps.     

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     

Improvement of full-thickness skin graft survival by application of vascular endothelial growth factor in rats

The effect of vascular endothelial growth factor (VEGF) on skin graft survival was studied in rats. Models of skin grafting on muscle and periosteum were designed. For the study of skin grafting on muscle model, 32 rats were divided into 4 groups. VEGF was administrated systemically after skin graft placement intrafascially injected into the recipient bed at the time of graft placement and topically applied to the recipient bed at the time of graft placement. Control groups consisted of grafts placed on sites without systemic or local VEGF treatment. With the study of skin grafting on periosteum, 40 rats were divided into 4 groups. VEGF was systemically, intraperiosteally, and topically applied to the recipient bed. The control animals received no treatment. On the fifth postoperative day, the survival area of each skin graft was measured and the graft was harvested for histology with CD31 immunohistochemical staining. The results showed that in skin grafting on muscle model, the mean viable percentage of the skin graft was 66.1% +/- 10.2% in the group receiving systemic application of VEGF and 56.1% +/- 9.8% in the group receiving VEGF intrafascia injection. The survival percentages were significantly higher than those found in the control group (22.5% +/- 7.7%) and the group with VEGF topical application (30.8% +/- 4.1%). In skin grafting on periosteum, the group receiving VEGF intraperiosteum injection reached a survival percentage of 50.5% +/- 4.3%, significantly higher than the groups with VEGF systemic application (28.7 +/- 5.5%), VEGF topical application (32.5% +/- 4.8%), and the control (25.8% +/- 6.0%). Histology showed that sections taken from grafts with VEGF intrafascia and intraperiosteum treatment revealed angiogenesis. The data demonstrated that administration of VEGF into the either muscular or periosteal recipient beds for skin grafting can improve the skin graft survival.     

Intradermal injection of GFP-producing adipose stromal cells promotes survival of random-pattern skin flaps in rats

Background

Tissue necrosis is a common complication in operations that use skin flaps for reconstructive surgery. Here we demonstrate the beneficial effect of autologous genetically modified adipose-derived stromal cells (ASCs) in the survival of random-pattern skin flaps.

Methods

ASCs were isolated from the inguinal fat pad of Wistar rats and genetically modified in order to permanently produce green fluorescent protein (GFP) using the Sleeping Beauty transposon technology. Autologous GFP-producing cells were then injected intradermally into random-pattern skin flaps planned on the dorsal area of rats.

Results

Injection of ASCs resulted in significant improvement of skin flap survival. Histological analysis showed that the connective tissue was almost intact in skin flaps treated with ASCs in contrast to disorganized tissues from mock-treated skin flaps. GFP ASCs were detected in the endothelium of blood vessels co-expressing the endothelial marker von Willebrand factor, thus suggesting that they promote blood vessel regeneration.

Conclusions

These findings indicate that transplantation of autologous GFP ASCs improve survival of skin flaps. This methodology suggests that the use of genetically modified ASCs producing, e.g., angiogenic factors may facilitate survival and integration of flaps in plastic surgery.     

Efficacy of topical nitroglycerin and transcutaneous electrical nerve stimulation on survival of random-pattern skin flaps in rats.

We compared the efficacy of topical nitroglycerin and transcutaneous electrical nerve stimulation (TENS) on the survival of random-pattern skin flaps in rats. Thirty Wistar albino rats were used and a dorsal, cranially-based random-pattern flap was raised. The rats were divided into three groups of 10 rats each. The first group had only the flap raised while the second and third groups were given topical nitroglycerin 5 mg or TENS for one hour a day for seven days. The amount of flap necrosis was measured on the seventh postoperative day. The mean area of necrosis in the flaps were 726.2, 544.2, and 150.0 mm2 in the control, nitroglycerin, and TENS groups, respectively. The mean percentage of flaps that necrosed were 51.9, 38.9, and 10.7 in the corresponding groups. The TENS group had significantly higher percentage area of flap surviving than the control (p < 0.0001) and nitroglycerin groups (p = 0.002). TENS, with its efficacy on survival and with negligible side-effects, could be a reliable treatment. Clinically, it can easily be used postoperatively when flaps become ischaemic, and will be tolerated well by patients.     

跨区供血皮瓣成活过程中血管构筑及内源性VEGF变化的实验研究

目的：探索跨区供血皮瓣的成活机制。方法：以大鼠旋髂深动脉为蒂制作包含肋间后动脉支配区域的右侧背部矩形跨区供血皮瓣，分别于术后即刻，术后1，2，3，5，7天时，观察皮瓣成活情况，成活过程中血管构筑及蒂部血管口径的改变以及内源性血管内皮细胞生长因子（Endogenous Vascular Endothelial Growth Factor，VEGF）免疫组化染色（取材部位为两血管支配区域中间血管网吻合部）。结果：皮瓣完全成活率100％。轴心动脉经吻合支向远端供血，经静脉吻合支回流。血管吻合区以远的血流方向与术前相反。术后即刻VEGF免疫组化染色阴性；1天后血管吻合支与术前相比明显增多、增粗，蒂部血管口径明显增粗，VEGF免疫组化染色阳性区域较多；3天时上述指标达高峰；7天时形成与顺流皮瓣相似的轴心血管，蒂部血管口径有所下降，VEGF免疫组化染色阴性。结论：跨区供血皮瓣的成活机制为血管的口径增大及数量增多，最终形成轴形血管皮瓣。内源性VEGF对皮瓣的成活起重要作用。     

pcDNA3.1(+)载体携带血管内皮生长因子治疗大鼠缺血皮瓣

目的 观察注射真核表达载体pcDNA3.1(+)携带血管内皮生长因子(VEGF)基因后对大鼠缺血皮瓣存活的影响.方法 构建重组质粒pcDNA3.1(+)-VEGF,将SD大鼠随机分成3组,每组10只.pcDNA3.1(+)-VEGF组:于背部皮肤皮下多点注射pcDNA3.1(+)-VEGF 30 μg/100μl;VEGF组:多点注射VEGF蛋白100 ng/100μl;生理盐水(NS)组:注射生理盐水100μl,注射2d后在其背部形成7 cm×3 cm的全厚随意型皮瓣,48 h后按原设计掀起皮瓣并原位缝合.术后10 d测量皮瓣的成活面积,计算成活面积百分比,并行免疫组织化学检测.结果 皮瓣成活面积百分比:pcDNA3.1(+)-VEGF组为(79.1±3.5)％,VEGF组为(62.2±2.7)％,NS组为(59.9±3.1)％,pcDNA3.1(+)-VEGF组皮瓣成活面积明显高于其他两组(P＜0.05).结论 皮下注射pcDNA3.1(+ )-VEGF可促进新生血管的形成,比单纯注射VEGF蛋白更能提高皮瓣的成活率.     

Adenoviral-mediated transfer of vascular endothelial growth factor 121 cDNA enhances myocardial perfusion and exercise performance in the nonischemic state

BACKGROUND: Angiogenic gene therapy has been demonstrated to enhance perfusion to ischemic tissues, but it is unknown whether the administration of angiogenic growth factors will increase blood flow to nonischemic tissues. This study investigates whether enhanced myocardial perfusion can be mediated by adenovirus-mediated transfer of vascular endothelial growth factor 121 cDNA to nonischemic myocardium. METHODS: New Zealand White rabbits received adenovirus (5 x 10(10) particle units) encoding for vascular endothelial growth factor 121 (n = 14) or a control vector without a transgene (n = 13) or saline solution (n = 9) via direct myocardial injection. Fluorescent microsphere perfusion studies and histologic analyses were performed 4 weeks later. In a parallel study, exercise treadmill testing was performed to assess the functional effects of this therapy in Sprague-Dawley rats. RESULTS: Microsphere assessment of myocardial perfusion in rabbits 4 weeks after adenovirus-encoding vascular endothelial growth factor administration was greater than that for rats injected with control vector without a transgene or saline solution (3.2 +/- 0.5 vs 2.7 +/- 0.7 and 2.4 +/- 0.4, respectively; P <.03). The endothelial cell count per high power field was increased in animals injected with adenovirus-encoding vascular endothelial growth factor versus animals injected with control vector without a transgene or saline solution (147 +/- 27 vs 123 +/- 14 and 125 +/- 16 cells, respectively), although this did not reach statistical significance (P =.12). Rats treated with adenovirus-encoding vascular endothelial growth factor also demonstrated prolonged exercise tolerance compared with rats injected with control vector without a transgene or saline solution (exhaustion time: 26 +/- 5 minutes vs 19 +/- 2 minutes and 20 +/- 3 minutes, respectively; P =.006). CONCLUSIONS: Adenovirus encoding-mediated transfer of vascular endothelial growth factor 121 induces an enhancement in regional perfusion in nonischemic myocardium that corresponds to changes in exercise tolerance. Adenovirus-encoding vascular endothelial growth factor therapy may be useful for inducing angiogenesis in the nonischemic state, such as for prophylactic therapy of early coronary artery disease.     

Transplantation of endothelial progenitor cells transferred by vascular endothelial growth factor gene for vascular regeneration of ischemic flaps

BACKGROUND: Neovascularization occurs through two mechanisms: angiogenesis and vasculogenesis. Therefore, there are two strategies to promote neovascularization: therapeutic angiogenesis and therapeutic vasculogenesis (endothelial progenitor cells therapy). MATERIALS AND METHODS: In this study, we examined whether or not endothelial progenitor cells combined with vascular endothelial growth factor (VEGF) gene therapy is useful for ischemia surgical flaps in vivo. At the same time, we quantitatively compared the neovascularization ability of transplanted endothelial progenitor cells (EPCs) transducted with VEGF165 gene and EPCs alone. EPCs were isolated from cord blood of healthy human volunteers, cultured in vitro for 7 days and identified by immunofluorescence. After transduced with VEGF165 gene in vitro, proliferative activity of EPCs was assessed using MTT assay. CM-DiI was used to trace EPCs in vivo 4 days after injection of 5 x 10(5) VEGF-transduced EPCs(VEGF-transduced EPCs group, n = 10), 5 x 10(5) EPCs (non-transduced EPCs group, n = 10) in 500 microL EBM-2 media, or 500 microL EBM-2 media (EBM-2 media group, n = 10) local, a cranially based flap was elevated on the back of nude mice. The percent flap survival, neovasculariztion and blood flow recovery of flaps was detected. RESULTS: EPCs expressed cell markers CD34, KDR, and CD133. A statistically significant increase in percent flap survival was observed in mice of VEGF-transduced EPCs group as compared with that of non-transduced EPCs group: 67.99 +/- 6.64% versus 59.43 +/- 4.69% (P < 0.01), and 41.24 +/- 2.44% in EBM-2 media group (P < 0.01). The capillary density and blood flow recovery of flaps in VEGF-transduced EPCs group were both improved. CM-DiI-labeled VEGF-transduced EPCs were observed in vivo and the numbers of cells increased. CONCLUSION: EPCs from human cord blood can increased neovascularization of ischemic flaps and augmented the survival areas, and VEGF-transduced EPCs have more powerful ability of promoting neovascularization in animal model of ischemic flaps.     

Improvement of venous flap survival by application of vascular endothelial growth factor in a rat model

Expression of vascular endothelial growth factor (VEGF) in the venous flap and the effect of exogenous VEGF on survival of the venous flap were studied in rats. A 4- x 4-cm groin type 2 venous skin flap was used in the study. In part 1, biopsies were taken from the flap at 0, 6, 12, 24, and 48 hours after the flaps were raised. VEGF gene expression was measured. In part 2, exogenous VEGF (1 microg/mL) was injected subdermally into the flaps in 10 rats before the flaps were replaced. Flaps that received a saline injection were used as the control. Skin paddle survival was measured on postoperative day 7. The results showed that VEGF expression was significantly increased at 24 and 48 hours after venous flap elevation (P < 0.05). Injection of exogenous VEGF to the flap significantly improved survival of the flap (73% of the flap) when compared with the control, which had a 39% mean percent survival (P < 0.05). We conclude that VEGF expression was increased in the venous flap. Administration of exogenous VEGF significantly improved survival of the venous flap.     

Effects of clopidogrel on survival of rat skin flaps.

Clopidogrel is a thienopyridine derivative that is chemically related to ticlopidine, which irreversibly inhibits platelet aggregation by selectively binding to adenylate cyclase-coupled adenosine diphosphate receptors on the platelet's surface. In animal models, clopidogrel has been shown to reduce the incidence of both arterial and venous thrombi. In the present study the effects of clopidogrel on the survival of rat epigastric island flaps was researched. Epigastric island flaps of 7x7cm were raised from symphisis pubis to arcus costa with the panniculus carnosus. The experimental group received seven doses of 25mg/kg clopidogrel postoperatively, the first dose given immediately after the suturing of the flaps. The rats were anaesthetised on postoperative day 7 to assess the survival of flaps. The difference between the clopidogrel and the control group was significant (P<0.005). The full-thickness skin samples obtained after the calculation of survival percentages revealed thinning of the epidermis layer and active chronic inflammation in both groups. However, diffuse dilated vessels, extravasated eritrocytes were seen in the clopidogrel group flaps. The results indicated a significant increase in flap survival in rats given clopidogrel. Further research is needed to assess the critical doses of clopidogrel to create optimal flap survival improvement.     

血管内皮生长因子基因治疗大鼠缺血皮瓣的实验研究

目的　研究血管内皮生长因子 (VEGF)基因治疗对大鼠缺血皮瓣生存的影响。方法建立大鼠缺血皮瓣的动物模型 ,采用直接注射法转移 pcD2 /hVEGF12 1真核表达质粒于大鼠缺血皮瓣肉膜层 ,术后 7d ,应用苏木素 伊红 (HE)染色、单光子发射计算机断层摄影 (SPECT)及计算机图像分析软件等方法检测皮下血管密度、皮瓣血供和皮瓣成活率。结果　经VEGF基因治疗组的大鼠缺血皮瓣与对照组相比较具有皮下血管密度增加、血液供应增多和皮瓣成活率显著性增高 (P <0 .0 1)。结论　VEGF基因治疗能够诱导新血管形成、增加血流灌注 ,促进缺血皮瓣的生存。