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.     

Fasciocutaneous flaps: an experimental model in the pig

No experimental studies have substantiated the claim that fasciocutaneous flaps are superior to skin flaps. Using fasciocutaneous flaps designed in the pig, both flap survival and blood flow were assessed. The forelimb and hindlimb fasciocutaneous flaps survived to 8.2 +/- 0.3 cm and 7.9 +/- 0.3 cm, respectively, compared with 7.3 +/- 0.3 cm and 6.7 +/- 0.3 cm for the comparable cutaneous flaps, a statistically significant finding (p less than 0.01). Random fasciocutaneous flaps survive 12 to 18 percent longer than skin flaps. Using the radioactive microsphere technique, blood flow was measured after flap elevation, and flap survival was estimated using fluorescein. Again, a significant difference in flap survival was found, but there was no significant difference in measured blood flow. This can be explained by the relatively large interval between blood flow measurements (2 cm) compared with the observed difference in survival length (1.0 +/- 0.3 cm).     

A study of the pharmacologic control of blood flow to acute skin flaps using xenon washout. Part I

This study was undertaken to understand the control mechanisms differentiating circulation to normal skin and acute skin flaps. The approach was to compare the effects of systemic vasoactive drugs on skin blood flow in rats in acute skin flaps and identical areas of control skin. With this model it was felt that systemic changes would affect both areas equally and any difference in response would be due to vascular control mechanisms unique to the flap. Xenon washout by percutaneous injection was chosen to measure blood flow. The results of over 8000 observations in these studies were: 1. Vasodilation enhances blood flow and flap survival. 2. Vasoconstriction decreases blood flow. 3. Depletion of sympathetic nerve terminals enhances blood flow and flap survival. 4. The acute flap is less sensitive to systemic alpha-agonists than control skin. 5. The acute flap is less sensitive to vasodilators acting at the receptor-site level than control skin. 6. Total sympathetic denervation does not occur. 7. Biologic increases in area of flap survival did occur in drug dose ranges predicted by xenon washout measurements in this model. These findings indicate that the vessels in an acutely raised skin flap have a greater vasospastic tone than is optimal for maximum nutrient blood flow. One explanation consistent with these findings is offered in which the mechanism responsible for this tone is the release of catecholamines from the sympathetic nerve terminals after the flap has been raised.     

Local pharmacological preconditioning increases the survival of experimental skin flaps in rats

Skin flap survival is dependent on an adequate blood supply and on the resistance to ischemia. Experimentally we tested the effect of pharmacological preconditioning on the survival of skin flaps. The survival of an ischemic skin flap (McFarlane flap) was tested using the local application of cobalt gel in three groups of Wistar rats. The mean flap necrosis of the control group was 59.15%. The rats treated by the cobalt gel once a week showed mean necrosis in 39.09%; the rats receiving the application of the cobalt gel three times a week in 26.33%. The treated groups presented with significantly lower flap necrosis in comparison to the untreated controls. There was a significant difference in the flap necrosis occurrence according to the application frequency of the cobalt gel. The expression of the genes involved in angiogenetic processes encoding vascular endothelial growth factor (VEGF) and glycolytic enzymes was influenced in a non-mitochondrial way in this study. The results show that non-mitochondrial preconditioning could prolong the survival of an ischemic flap.     

Early circulatory and metabolic events in island skin flaps of the pig

Circulatory and metabolic skin-flap events were studied prior to and up to 6 hours after elevation of buttock island flaps in pigs. During the elevation, significant reductions in superficial skin blood flow, measured by laser Doppler flowmetry (LDF) and dermal flap temperature, were seen. Significant correlations were found between blood flow and temperature. Total flap blood flow, measured as venous outflow, also showed an initial transient decrease, but 2 hours after flap construction, venous outflow had returned to preoperative values. A significant increase in lactate release, together with increased oxygen consumption and glucose uptake, was seen 4 hours after the surgical intervention. Hypoxanthine release, indicating ischemia, was seen only during the first hour after flap elevation. Noradrenaline outflow was noted after 4 and 6 hours, but there was no parallel reduction in flap blood flow. A great deal of the flow reduction in acutely elevated island flaps may thus be due to primary hypothermia rather than to the degenerative release of noradrenaline, which seems to have no early effect on skin flap blood flow. On the other hand, the noradrenaline release may be linked to an increased metabolic activity in the skin flaps.     

A study on the effectiveness of a thromboxane synthetase inhibitor (OKY-046) in increasing the survival length of skin flaps

In an experimental study to test the thromboxane (TX) synthetase inhibitor OKY-046, two random-pattern skin flaps, each measuring 15.5 x 2 cm, and caudally based, were elevated on the backs of rabbits, and the effect of the test drug on their survival length was evaluated. The results indicated that the survival length of the skin flaps was 4.5 +/- 0.2 cm in the control group and 6.8 +/- 0.3 cm in the OKY-046-treated group, hence exceeding the control value by more than 50 percent, which was statistically significant. A laser speckle flow-meter showed that the OKY-046-treated flaps had significantly greater blood flow as compared with the control group both at 1 and 48 hours after operation. Whereas the blood flow values were significantly lower at 48 hours than at 1 hour after operation in the control group, no such reduction was noted in the OKY-046-treated group. On the other hand, while plasma TXB2 was found elevated at 1 hour postoperatively in the control group, such a response to the surgical intervention was blocked and the plasma TXB2/6-keto prostaglandin (PG) F1a ratio was decreased in the OKY-046-treated group. These results clearly indicated that OKY-046 suppressed a plasma thromboxane elevation induced by surgery, it augmented the flap blood flow, and it thereby increased flap survival length, suggesting that the drug might be helpful clinically and that further investigation must be carried out concerning its application.     

Effects of sodium pentobarbital anesthesia on blood flow in skin, myocutaneous, and fasciocutaneous flaps in swine.

Drug effect on flap blood flow is most commonly determined in anesthetized animals, yet the effect of the anesthetic is often poorly understood. Halothane and nitrous oxide cause profound changes in skin blood flow and thus provide an unsuitable anesthetic technique for use in measuring drug effects on skin and myocutaneous flaps in swine. The goal of this study was to determine the effects of sodium pentobarbital anesthesia on cardiovascular parameters and blood flow in skin, myocutaneous, and fasciocutaneous flaps in pigs. In seven pigs, 7 forelimb skin flaps, 7 forelimb fasciocutaneous flaps, 14 arterial buttock flaps, and 14 latissimus dorsi flaps were created. Blood flow was measured at 2-cm intervals along each flap while the animal was awake and anesthetized. A cardiac depressant effect of pentobarbital was observed, but no change in blood flow could be demonstrated in control skin or control muscle. However, pentobarbital did significantly increase blood flow in all viable portions of arterial and random skin flaps, fasciocutaneous flaps, and the cutaneous segments of the latissimus dorsi flap. These demonstrated effects of pentobarbital should be taken into consideration in designing and analyzing studies of flap blood flow in the acute postoperative phase.     

Systemic Preconditioning by a Prolyl Hydroxylase Inhibitor Promotes Prevention of Skin Flap Necrosis via HIF-1-Induced Bone Marrow-Derived Cells

Background

Local skin flaps often present with flap necrosis caused by critical disruption of the blood supply. Although animal studies demonstrate enhanced angiogenesis in ischemic tissue, no strategy for clinical application of this phenomenon has yet been defined. Hypoxia-inducible factor 1 (HIF-1) plays a pivotal role in ischemic vascular responses, and its expression is induced by the prolyl hydroxylase inhibitor dimethyloxalylglycine (DMOG). We assessed whether preoperative stabilization of HIF-1 by systemic introduction of DMOG improves skin flap survival.

Methods and Results

Mice with ischemic skin flaps on the dorsum were treated intraperitoneally with DMOG 48 hr prior to surgery. The surviving area with neovascularization of the ischemic flaps was significantly greater in the DMOG-treated mice. Significantly fewer apoptotic cells were present in the ischemic flaps of DMOG-treated mice. Interestingly, marked increases in circulating endothelial progenitor cells (EPCs) and bone marrow proliferative progenitor cells were observed within 48 hr after DMOG treatment. Furthermore, heterozygous HIF-1α-deficient mice exhibited smaller surviving flap areas, fewer circulating EPCs, and larger numbers of apoptotic cells than did wild-type mice, while DMOG pretreatment of the mutant mice completely restored these parameters. Finally, reconstitution of wild-type mice with the heterozygous deficient bone marrow cells significantly decreased skin flap survival.

Conclusion

We demonstrated that transient activation of the HIF signaling pathway by a single systemic DMOG treatment upregulates not only anti-apoptotic pathways but also enhances neovascularization with concomitant increase in the numbers of bone marrow-derived progenitor cells.     

Angiogenic and anti-inflammatory properties of azadirachtin A improve random skin flap survival in rats

Random skin flaps are widely used to repair tissue defects. However, the distal flap regions are prone to ischemic necrosis, limiting clinical applications. Azadirachtin A, a fruit extract from the neem, improves tissue blood supply and metabolism, reduces cell swelling, promotes tissue healing, and prevents venous thrombosis. We explored whether it enhances random skin flap survival. Fifty-four Sprague-Dawley rats were divided into control, low-dose, and high-dose Azadirachtin A-treated groups using a random number table. We used an improved version of the McFarlane technique to create flaps. On day 2, superoxide dismutase and malondialdehyde levels were measured. Tissue slices prepared on day 7 were stained with hematoxylin and eosin. The expression levels of vascular endothelial growth factor (VEGF), toll-like receptor 4 (TLR4), nuclear factor kappa-B (NF-kB), interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) were immunohistochemically assayed. Microcirculatory blood flow was measured via laser Doppler blood flowmetry. Flap angiography was performed using the lead-oxide gelatin injection technique. And the azadirachtin A groups exhibited a greater mean flap survival area, an improved mean blood vessel density, a greater blood flow, and higher superoxide dismutase and VEGF levels, especially at the high dose. Azadirachtin A markedly reduced the levels of TNF-α, IL-6, IL-1β, TLR4, and NF-kB. These findings suggest that azadirachtin A promotes random skin flap survival by improving the blood supply, reducing tissue inflammation, and inhibiting flap ischemia reperfusion injury.     

Surgical augmentation of skin blood flow and viability in a pig musculocutaneous flap model

A porcine rectus abdominis musculocutaneous (TRAM) flap model was designed and validated in nine pigs. This TRAM flap was based on the deep inferior epigastric (DIE) vessels with an 8 x 18 cm transverse skin paddle at the superior end of the rectus abdominis muscle. The model was subsequently used to test our hypothesis of surgical augmentation of flap viability by vascular territory expansion. Specifically, we observed that ligation of the superior epigastric (SE) vessels at 4, 7, 14, and 28 days (N = 6 to 8) prior to raising the TRAM flaps significantly increased (p less than 0.05) the length and area of the viable skin in the transverse skin paddles of the treatment flaps compared with the contralateral shammanipulated control flaps. This significant increase in skin viability was seen to be accompanied by a significant increase (p less than 0.05) in skin and muscle capillary blood flow in the treatment TRAM flaps compared with the controls (N = 9). The mechanism of vascular territory expansion is unclear. We postulate that hypoxia resulting from the ligation of the superior epigastric vessels prior to the flap surgery may play a role in the triggering of the deep inferior epigastric artery to take over some of the territory previously perfused by the superior epigastric artery. This would then increase the skin and muscle capillary blood flow in the transverse paddle when the TRAM flap was raised on the deep inferior epigastric vascular pedicle.     

Cyclophosphamide-induced neutropenia: effect on postischemic skin-flap survival.

In a blinded study, 24 pigs were randomized to a 5-day preoperative treatment regimen of cyclophosphamide (n = 12) or placebo (n = 12). At operation, buttock cutaneous and latissimus dorsi myocutaneous flaps were created and then subjected to 6 hours of global ischemia. After 24 hours of reperfusion, flap skin and muscle survivals were determined. All cyclophosphamide-treated animals were rendered neutropenic (less than 500 neutrophils/mm3 of peripheral blood). The results show that neutropenia had no effect on postischemic buttock cutaneous flap survival. In contrast, cyclophosphamide-induced neutropenia demonstrated a significant protective effect on postischemic latissimus dorsi myocutaneous flap survival. This study further implicates the neutrophil as a significant factor in the mediation of ischemia/reperfusion injury of myocutaneous flaps.     

Experimental studies in the survival of venous island flaps without arterial inflow

The authors have studied the effects of various circulatory settings on flap survival. The dog model was used to study the survival of venous flaps without arterial inflow both as island and free flaps. Venous flaps were compared with arterial flaps without venous outflow and standard island flaps with arterial inflow and venous outflow. Attempts were made to study their vascular morphology and blood gas changes. The venous flaps without arterial inflow survived with normal hair growth and wound healing, as did the standard island flaps. These observations suggest that capillary diffusion can occur without the continuous flow of blood through a capillary. Several possible mechanisms to explain survival of the venous flaps without arterial inflow were discussed. These observations could be important in providing an animal model to study microcirculation and a possible new area for microsurgical transfer of a skin flap.     

Measurement of skin blood flow in delayed deltopectoral flaps, using local clearance of 133Xenon.

A study was made to determine the skin blood flow at the deltoid region in 89 cases, and the regional blood flow of delayed deltopectoral flaps, using the local clearance of 133Xe. The change in the skin blood flow, before and after a delay procedure of the deltopectoral flap, was measured in 27 patients--and the following results were obtained. (1) There was a linear tendency to a decreasing flow, one found to be statistically significant, with increasing age of the patient. (2) A significant correlation was found between the skin blood flow and the blood flow of the subcutaneous tissue. (3) The blood flow after we raised one side of a deltopectoral flap and lined it with a split-skin graft was higher than that found after a U-shaped undermining and not lining a flap. (4) The rate of successful transfer of a deltopectoral flap was found to be low when the 133Xe clearance rate was less than 0.07.     

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.     

Experimental pretransfer expansion of free-flap donor sites: I. Flap viability and expansion characteristics

Expansion of cutaneous and myocutaneous free-flap donor sites prior to elevation is possible in the pig model. There is no significant difference in survival between control and expanded cutaneous buttock and myocutaneous latissimus dorsi flaps after elevation solely on their axial pedicles. Axial-pattern flap expansion appears to augment capillary blood flow. The maximum amount of expansion occurs directly over the center of the expander and decreases toward the periphery. There is virtually no expansion of skin adjacent to the expander.     

The Effect of Local Application of Natural Hirudin on Random Pattern Skin Flap Microcirculation in a Porcine Model

The aim of this study was to investigate the effect of local administration of hirudin in improving random pattern skin flap microcirculation in a porcine model. Five Chinese minipigs were used and six dorsal random pattern skin flaps were elevated in each animal (4 × 14 cm). All flaps (n = 30) were assigned to experimental (n = 10), control (n = 10), and sham (n = 10) groups. Flap edema measurement showed that edema in experimental flaps was more severe (P < 0.05) than either control or sham flaps. Local blood flow detection showed an increased image signal of blood flow in experimental flaps instead of an obvious avascular area in control and sham flaps. The survival area was significantly greater in experimental group (67.6 ± 2.1 %) as compared to control (45.2 ± 1.4 %) or sham (48.3 ± 1.1 %) group (P < 0.05). Our data showed that local administration of hirudin can significantly improve random pattern skin flap microcirculation in over dimensioned random pattern skin flaps in a porcine model.     

HGF基因真核表达质粒的构建及其细胞表达

目的:构建真核表达质粒p EGFP-N1-HGF并观察干细胞生长因子(hepatocyte growth factor,HGF)在人皮肤成纤维中的表达。方法:从扩增人的间充质干细胞中提取全长RNA为模板,设计合成引物,用RT-PCR法扩增出HGF基因片段,然后将HGF基因片段连接于真核表达质粒p EGFP-N1中。双酶切鉴定及测序分析后,用脂质体包裹转染体外培养的人的皮肤成纤维细胞,通过荧光显微镜观察其转染效果,酶联免疫吸附测定法(enzyme-linked immunosorbent assay,ELISA)检测HGF蛋白的表达。结果:经酶切鉴定及测序验证p EGFP-N1-HGF构建正确,经转染的人皮肤纤维细胞中观察到较强的绿色荧光并且可以表达一定量的HGF。结论:成功构建了真核表达质粒p EGFP-N1-HGF,为基因治疗脱发疾病提供实验基础。     

Dermofluorometry: thresholds for predicting flap survival

The dye fluorescence index (DFI) has been cited as an accurate predictor of skin-flap survival. However, two thresholds, one each for flap survival and flap necrosis, have been advocated. A DFI of less than 15 to 20 percent predicts failure, and a DFI greater than 35 to 50 percent predicts survival. Values of 20 to 35 percent indicate an uncertain outcome. The present study was undertaken (1) to determine the optimum threshold for flap survival prediction in pigs, and (2) to compare dermofluorometry with flap blood flow as measured by radioactive microspheres. Dermofluorometry was found to be an accurate (90 percent) and repeatable predictor of skin and fasciocutaneous flap survival in pigs. At 2 and 5 hours after flap elevation, the optimum DFI thresholds are 7 and 27 percent, respectively. This reflects the dynamic nature of circulation in acute skin flaps and the increased dye delivery over time. Using these calculated thresholds, a high degree of correlation was found with survival estimated at 24 hours. Dermofluorometry also was correlated with the blood flow index. Thus not only is it an accurate flap monitor, but a quantitative estimate of flap blood flow can be obtained.     

Hydrogen clearance: assessment of technique for measurement of skin-flap blood flow in pigs

The hydrogen clearance technique has been used for many years by investigators to determine brain blood flow and has been partially validated in this setting using other methods of blood flow measurement. The method has been modified to allow blood flow measurements in skin, but the accuracy of H2 clearance for measuring skin blood flow has not been determined. Multiple blood flow measurements were performed using H2 clearance and radioactive microspheres on skin flaps and control skin in pigs. On 12 pigs, a total of 117 flap and 42 control skin measurements were available for analysis. There was no significant difference between the two techniques in measuring mean control skin blood flow. In skin flaps, H2 clearance was significantly correlated to microsphere-measured blood flow, but it consistently gave an overestimate. Sources of error may include injury to the tissues by insertion of electrodes, consumption of H2 by the electrodes, or diffusion of H2 from the relatively ischemic flap to its well-vascularized bed. Further studies are necessary to determine the cause of this error and to measure the technique's accuracy in skeletal muscle and other flaps.     

The effect of autologous endothelial progenitor cell transplantation combined with extracorporeal shock-wave therapy on ischemic skin flaps in rats

BackgroundEndothelial progenitor cells (EPCs) have been used to revascularize ischemic tissues, but only limited effect can be achieved. Extracorporeal shock-wave therapy (ESWT) is a promising angiogenic strategy. We hypothesized that EPC transplantation combined with ESWT would greatly benefit the survival of ischemic skin flaps.MethodsSixty-four male Sprague-Dawley rats were divided into 4 groups (n = 16 in each group): group 1 (serving as sham control), group 2 (treated with subcutaneous EPC implantation, 1.0 × 10⁶ cells), group 3 (treated with ESWT, 300 impulses at 0.10 mJ/mm²) and group 4 (treated with EPCs implantation combined with ESWT). Ischemic skin flaps were made on the backs of rats and treated accordingly. Blood flow of skin flaps was measured periodically after operation, and flap survival rates were compared. Tissue samples were harvested at 2 weeks postoperatively from each group.ResultsThe survival rate of skin flaps in group 4 was 87.5 ± 10.23%, which was statistically significantly higher than other groups. Histologic examination showed that the capillary density was higher in the dual-treatment group than in the two single-treatment groups. Compared with groups 2 and 3, blood perfusion increased significantly in group 4. A drastic increase of vWF+ cells was observed in the ischemic skin flaps on immunofluorescence staining in group 4. The expressions of chemotactic factors and angiogenic factors were higher in group 4.ConclusionsCombined treatment with EPCs and ESWT is superior to either EPCs or ESWT alone in improving the survival of ischemic skin flaps in rats.