Augmentation of blood flow in delayed random skin flaps in the pig: effect of length of delay period and angiogenesis

Skin capillary blood flow and angiogenesis were studied by radioactive microsphere and morphometry technique, respectively, in delayed random skin flaps in the pig. Skin flaps were delayed for 2, 3, 4, 6, or 14 days. Blood flow was measured 6 hours after complete raising of acute and delayed random skin flaps on the opposite flanks of the same pig. It was observed that the capillary blood flow increased significantly (p less than 0.05) within 2 days of delay compared to the acute skin flaps. This capillary blood flow further increased by about 100 percent between days 2 and 3, started to plateau after day 3, and remained unchanged between days 4 and 14 of delay. This increase in capillary blood flow was mainly in the distal portion of the delayed skin flaps. There was no indication of an increase in the density of arteries in all delay periods studied. Our observations did not support the hypotheses that the delay phenomenon involves angiogenesis or long-term adaptation to ischemia, as have been hypothesized previously. The possible mechanism of delay is discussed.     

Basic fibroblast growth factor expression following surgical delay of rat transverse rectus abdominis myocutaneous flaps

Partial transverse rectus abdominis myocutaneous (TRAM) flap loss in breast reconstruction can be a devastating complication for both patient and surgeon. Surgical delay of the TRAM flap has been shown to improve flap viability and has been advocated in "high-risk" patients seeking autogenous breast reconstruction. Despite extensive clinical evidence of the effectiveness of surgical delay of TRAM flaps, the mechanisms by which the delay phenomenon occurs remain poorly understood. To examine whether angiogenic growth factors such as basic fibroblast growth factor (bFGF) may play a role in the delay phenomenon, the authors studied the expression of bFGF in rat TRAM flaps subjected to surgical delay. Thirty-five female Sprague-Dawley rats were randomly assigned to one of four TRAM flap groups: no delay (n = 6), 7-day delay (n = 12), 14-day delay (n = 10), or 21-day delay (n = 7). Surgical delay consisted of incising skin around the perimeter of the planned 2.5 x 5.0-cm TRAM flap followed by ablation of both superior epigastric arteries and the left inferior epigastric artery, thus preserving the right inferior epigastric artery (the nondominant blood supply to the rectus abdominis muscle of the rat). TRAM flaps were then elevated after 7, 14, and 21 days of delay by raising zones II, III, and IV off the abdominal wall fascia. Once hemostasis was assured, the flaps were sutured back in place. All flaps were designed with the upper border of the flap 1 cm below the xiphoid tip. Three days after the TRAM procedure, postfluorescein planimetry was used to determine percent area viability of both superficial and deep portions of TRAM flaps. All rats were euthanized and full-thickness TRAM specimens were taken from zones I, II, III, and IV for enzyme-linked immunoabsorbent assay analysis of bFGF levels. Statistical testing was done by t test (percent viability) and two-way analysis of variance (bFGF levels). All delayed flaps had significantly higher bFGF levels when compared with all nondelayed control flaps (p < 0.05). The bFGF levels were not different in the rats that received TRAM flaps 7, 14, or 21 days after delay surgery. There was also no significant difference in bFGF levels among zones I through IV. Control rats had more peripheral zone necrosis compared with all delayed TRAM rats. All delayed flaps had a significantly higher area of flap viability superficially than nondelayed control flaps (p < 0.05). There was no difference in deep flap viability. Surgical delay of rat TRAM flaps is associated with improved flap viability and significantly elevated levels of bFGF over nondelayed TRAM flaps at postoperative day 3 after TRAM surgery. The increases in bFGF noted at this time point suggests that bFGF may play a role in the improved TRAM flap viability observed after delay surgery. Further investigation is needed to evaluate the role bFGF may play in the delay phenomenon.     

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.     

Increased survival and vascularity of random-pattern skin flaps elevated in controlled, expanded skin

Controlled clinical tissue expansion, a new technique of providing donor tissue, results in an increase in surface area of expanded skin. The aim of the present study was to determine the effect of controlled tissue expansion on the surviving lengths of random-pattern skin flaps elevated in expanded tissue. In five pigs the surviving lengths of flaps raised in skin expanded for 5 weeks using a 250-cc rectangular Radovan-type tissue expander were compared with the survival lengths of flaps elevated in tissue in which a similar prosthesis was not expanded, bipedicle flaps delayed for 5 weeks, and control acutely raised random-pattern flaps. The expanded flaps had a mean increase in surviving length of 117 percent over control flaps, which was statistically significant. The delay flaps had an increase in survival of 73 percent over control flaps, which was also statistically significant. There was no significant difference in survival between expanded flaps and delayed flaps. Morphologic studies using radiographic techniques on one pig demonstrated increased vascularity with tissue expansion. The results of this work demonstrate that in addition to providing increased surface area with controlled expansion, flaps raised in expanded skin have a significantly augmented surviving length. The mechanism for this increased vascularity with expansion is not known at this time, but it may be due to physical forces associated with expansion acting as a stimulus for angiogenesis.     

Tissue oxygen measurements in delayed skin flaps: a reconsideration of the mechanisms of the delay phenomenon

The delay phenomenon was studied by measuring tissue oxygen tension (PsqO2) for 3 weeks in delayed flaps and normal adjacent contralateral skin in seven mongrel dogs. The PsqO2 fell after elevation of a bipedicle flap and rose again to normal by day 14. Delivery of oxygen to this flap was improved by surgical delay, so that when the bipedicle flap was reelevated on day 14 and its distal pedicle divided, minimal changes in PsqO2 occurred. When the control area was elevated on day 14 as a random-pattern flap, it had higher PsqO2 values than measured in the bipedicle flap on day 0, and therefore, it too had participated in the delay phenomenon, even though only its midline edge had been incised. An anatomic explanation for the findings was sought in wounds made in 10 rabbit ear chambers. After injury, blood flow was seen to be rerouted parallel to the incision line and was increased first by vasodilation and then also by angiogenesis until about day 14. Rerouting of blood by injury, inflammation, and angiogenesis due to repair appears to account for a significant portion of the delay phenomenon.     

Effect of radiation on skin expansion and skin flap viability in pigs

The aim of the present study was to investigate the effect of radiation treatment both on skin tissue expansion with the chronic inflation of subcutaneous expanders and on skin flap viability in surgically delayed and expanded skin in the pig. One flank in each of six pigs (initially weighing 17 +/- 1.8 kg) was randomly assigned for radiation treatment, and the contralateral flank served as a nonirradiated control. Three mirror-image, 8 x 10 cm, rectangular templates were marked on each flank; these templates were randomly assigned to the construction of a delayed skin flap (group A), a skin flap raised on expanded skin (group B), or a skin flap raised on expanded skin with a capsulectomy before flap surgery (group C). Radiation treatment was performed using sequential radiation with three fractions per week (810 cGy/fraction) for 2 weeks, with a total dose of 4,860 cGy. Twelve weeks after radiation treatment, skin expanders (8 x 10 cm) were installed subcutaneously in the locations assigned for skin expansion. Skin expansion by the inflation of subcutaneous skin expanders with saline twice weekly was started 8 weeks later and lasted for 3 weeks. Two weeks after surgical delay and the last skin expansion, 8 x 20 cm skin flaps were raised on the locations assigned for delayed skin flaps, expanded skin flaps, and expanded skin flaps with a capsulectomy. Skin flap viability was assessed 24 hours later using a fluorescein dye-staining technique. Skin expansion by the inflation of subcutaneous expanders with saline was slower (p < 0.05) in the radiated skin (39 +/- 6 ml/filling) than in the nonirradiated control skin (51 +/- 6 ml/filling). Radiation reduced the overall area of expanded skin by 23 percent (p < 0.05) compared with the control. Radiation treatment also reduced skin viability by 36 percent (p < 0.05) in the delayed skin flaps, 27 percent (p = 0.10) in the expanded skin flaps, and 36 percent (p < 0.05) in the expanded skin flaps with a capsulectomy when compared with their contralateral, nonirradiated controls. There were no significant differences in skin viability among these three types of skin flaps within the radiated and nonirradiated groups. Taken together, these observations indicate that radiation treatment reduced the effectiveness of the surgical delay procedure, the amount of subcutaneous skin expansion (by an increase in skin area), and skin flap viability. However, a capsulectomy alone did not affect the viability of skin flaps raised on expanded skin.     

Pathogenesis of ischemic necrosis in random-pattern skin flaps induced by long-term low-dose nicotine treatment in the rat

The objectives of the present experiments were to study the effects of long-term low-dose nicotine treatment on skin hemodynamics, viability, and microvascular morphology in 4 x 10 cm dorsally based acute random-pattern skin flaps in the rat. In addition, the reversibility of the nicotine-induced detrimental effects on skin-flap viability following cessation of nicotine treatment also was investigated. Low-dose nicotine (0.6 mg/kg) administered twice daily and subcutaneously for 24 weeks significantly (p less than 0.05) decreased skin-flap capillary blood flow, distal perfusion, and length and area of skin viability compared with the saline-treated control (n = 15). However, these same parameters in rats (n = 15) whose nicotine treatment had been withheld for 2 weeks prior to skin-flap surgery were not significantly different from the control, thus indicating that the detrimental effects of this long-term, low-dose nicotine treatment were reversible. The mean plasma level of nicotine in the nicotine-treated rats was 8.1 +/- 0.4 micrograms/dl and was within the range of plasma nicotine levels reported for human heavy cigarette smokers. Light and electron microscopic studies did not show evidence of histologic damage to the cutaneous microvasculature in acute random-pattern skin flaps and samples of normal (nonoperated) skin in nicotine-treated rats. It is concluded that long-term plasma levels of nicotine similar to those of heavy cigarette smokers are detrimental to the capillary blood flow and viability of random-pattern skin flaps in the rat. These deleterious effects can be avoided if skin flaps are raised 2 weeks after cessation of nicotine treatment. This low-dose nicotine treatment does not cause histologic damage to the microvasculature. Other pathogenic mechanisms of nicotine-induced skin flap ischemia are discussed.     

Clamp delay: an effective new method of nonsurgical delay

This study introduces an effective new method of nonsurgical delay. In this new method, a special clamp that compressed a bipedicled skin fold along the sides of a proposed flap was glued to rat dorsa. The study also used a control group of untreated flaps and a group of flaps delayed by the conventional surgical procedure involving conventional parallel incisions. Eight days later, 1 x 6 cm reverse McFarlane flaps were isolated from the wound and raised. After 5 days, the survival length of the flaps was measured. Viability of the flaps delayed by the clamps [40.5 +/- 2.0 mm (mean +/- standard error); n = 10] did not differ from that of surgically delayed flaps (41.3 +/- 3.6 mm; n = 8) and was significantly higher (p < 0.001) than the survival of control flaps (26.3 +/- 0.6 mm; n = 10). Clamp delay can be useful in flap research that explores the relative significance of the vessel obstruction and biochemical processes that follow the surgical delay procedure. This method also offers a new perspective by introducing the concept of nonsurgical delay into clinical practice.     

Nutrient blood flow in delayed axial pattern skin flaps in pigs.

Blood flow was investigated in 112 skin flaps (84 delayed and 28 undelayed) in 28 pigs. The flow was significantly (P less than 0.001) increased over the control flow with increasing delay intervals, reaching the greatest flow at one week after the delay (paralleling the increase in tissue survival). This increased blood flow persisted after definitive flap raising. The circulatory adjustments within the first week of the delay constitute the delay phenomenon, and they determine the ultimate viability of the skin flap in this model.     

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.     

Reducing the vascular delay period in latissimus dorsi muscle flaps for use in cardiomyoplasty

Although the mechanism by which vascular delay benefits skin flaps is not completely understood, this topic has been extensively studied and reported on in the literature. In contrast, little has been documented about the effects of vascular delay in skeletal muscle flaps. Recent animal studies tested the effectiveness of vascular delay to enhance latissimus dorsi muscle flap viability for use in cardiomyoplasty and found that it prevented distal flap necrosis. However, these studies did not define the optimal time period necessary to achieve this beneficial effect. The purpose of this study was to determine how many days of "delay" can elicit the beneficial effects of vascular delay on latissimus dorsi muscle flaps. To accomplish this, 90 latissimus dorsi muscles of 45 male Sprague-Dawley rats were randomly subjected to vascular delay on one side or a sham procedure on the other. After predetermined delay periods (0, 3, 7, 10, and 14 days) or a sham procedure, all latissimus dorsi muscles were elevated as single pedicled flaps based only on their thoracodorsal neurovascular pedicle. Latissimus dorsi muscle perfusion was measured using a Laser Doppler Perfusion Imager just before and immediately after flap elevation. The muscles were then returned to their original vascular beds, isolated from adjacent tissue with Silastic film, sutured into place to maintain their original size and shape, and left there for 5 days. After 5 days, the latissimus dorsi muscle flaps were dissected free, scanned again (Laser Doppler Perfusion Imager-perfusion measurements), and the area of distal necrosis was measured using digitized planimetry of magnified images. The authors' results showed that delay periods of 3, 7, 10, and 14 days significantly increased (p < 0.05) blood perfusion and decreased (p < 0.05) distal flap necrosis when compared with sham controls. On the basis of these findings, the authors conclude that in their rat latissimus dorsi muscle flap model the beneficial effects of vascular delay are present as early as 3 days. If these findings also hold true in humans, they could be useful in cardiomyoplasty by allowing surgeons to shorten the amount of time between the vascular delay procedure and the cardiomyoplasty procedure in these very sick patients.     

An anatomic review of the delay phenomenon: II. Clinical applications.

This paper applies the anatomic concepts and data obtained from our animal experimental studies of the delay phenomenon to a series of clinical cases. Similar clinical results were obtained to those seen in Part I of our study when skin flaps were raised with and without a delay, when a tissue expander was used, and when the delay technique was extended to musculocutaneous flaps. In each instance, the cutaneous perforators were identified with the Doppler probe to facilitate the delay of specific vessels rather than dividing those at random. Intraoperative arteriograms and venograms reveal that the choke arteries dilate and the anatomically unfavorable valved vein segments become regurgitant. The end result is the observation that at least one additional anatomic vascular territory can be added to the length of a flap with safety following a surgical delay.     

The effect of delay on flap survival in an irradiated field

Chronic radiation skin injury without ulceration was induced in rats by administering either 3000 or 5000 rads in staged doses. Either 4 or 8 months later, McFarlane et al. dorsal flaps were elevated, with half being delayed and half non-delayed. Measurements showed that flap survival in irradiated skin was significantly increased by delay [approximately two of four experimental groups (4 months, 5000 rads; and 8 months, 3000 rads)]; flap survival was increased with borderline significance in a third experimental group (8 months, 5000 rads). These data indicate that flap survival can be increased by delay in an irradiated field. The presence of the vascular delay phenomenon suggests that microvascular occlusion alone cannot account for radiation-induced complications in skin.     

Skin recycling following neovascularization using the rat musculocutaneous flap model.

The recycling of a skin territory as part of a musculocutaneous flap despite prior division of existing musculocutaneous perforators or vice versa within an axial cutaneous flap using skin from a previous musculocutaneous flap may sometimes be done safely if an adequate time period has been allotted to permit sufficient neovascularization from adjacent tissues. In order to test this clinically observed phenomenon, a musculocutaneous flap model based on perforators from the rat rectus abdominis muscle was developed and observed to have complete reliability. Groups of five Sprague-Dawley rats each were sequentially utilized to prove that by a single week following creation of a rectus abdominis musculocutaneous flap adequate peripheral neovascularization would evolve to permit total viability of secondary axial epigastric cutaneous flaps incorporating the same skin that initially was the cutaneous portion of the muscle flap. The converse was also confirmed possible, again using sequential groups of five rats each, in that by 2 weeks the skin of an initial abdominal cutaneous flap could instead be safely transposed and nourished as part of a rectus abdominis musculocutaneous flap. The proposition concerning the reliable reuse of identical skin territories as part of disparate metachronous flap configurations appears to be valid.     

Role of nitric oxide in skin flap delay

Surgical delay of skin flaps before transfer is known to improve flap viability. This study attempts to elucidate the mechanism of vasodilation by exploring the effects of nitric oxide on the microcirculation of delayed skin flaps. Using a skin flap model in 22 CD-1 white mice, the diameter of two nonterminal choke arteries was measured using in vivo videomicroscopy. Vessel flow was also measured using an optical Doppler velocimeter. Similar measurements were recorded in several animals on the same vessels in which subcutaneous dissection without elevation was performed. Average vessel diameter ranged from 21.77 to 25.55 microm before skin flap delay. Average flow ranged from 1.72 to 2.44 nl/sec before delay. Next, each animal received an intraperitoneal dose of nitro-aminomethyl-1-arginine (L-NAME), a nitric oxide synthase inhibitor delivered by means of osmotic pump at a level of 0 (n = 13 arteries), 20 (n = 10), 50 (n = 8), or 100 mg/kg/day (n = 7). Flaps were re-elevated 72 hours later and the aforementioned measurements were repeated. Vessel diameter increased to 44.92 microm in the control (0 mg/kg L-NAME) animals. Flow increased to 7.66 nl/sec in the control animals. Vessel dilation and flow did not change significantly in the nonoperative vessels. As the dose of L-NAME increased in the treated animals, there was a significant decrease in vasodilation and flow (p = 0.015 and p = 0.03, respectively). The authors' results demonstrate that nitric oxide is an important element of vasodilation and contributor to the phenomenon of skin flap delay.     

负压封闭引流技术在腹部带蒂皮瓣供瓣区的应用

目的:探讨负压封闭引流(vaccumsealingdrainage,VSD)技术在腹部带蒂皮瓣供瓣区中的治疗作用。方法:10例上肢深度创面大小为9×6 cm-14×9 cm,创面清创后行腹部带蒂皮瓣移植,皮瓣面积10×7 cm-20×15 cm,供瓣区应用VSD覆盖,医用半透性贴膜封闭整个术区,连接中心负压,持续低负压吸引,每日外用1000 mL生理盐水持续冲洗VSD。约3周后行皮瓣断蒂术,供瓣区植皮。结果:10例皮瓣全部存活,1例患者术后当天引流区域出现疼痛,经止痛治疗后未再出现。1例患者术后2周出现VSD堵塞,经处理后恢复正常引流。无VSD装置更换病例。供瓣区周边皮肤无浸渍及感染现象,VSD撤除后见基底肉芽组织生长新鲜,创面均无感染,移植皮片存活。结论:VSD技术可以有效保护供瓣区,刺激肉芽生长,为后期皮片移植闭合创面提供了良好条件。VSD技术可安全有效的应用于腹部带蒂皮瓣供瓣区。     

Postoperative irradiation after reconstructive surgery: comparative study of radiosensitivity between free-skin grafts and skin flaps

Radiation effects after reconstructive surgery (free-skin grafts and skin flaps) were studied in the rat, and the optimum time for irradiation was determined. The radiosensitivity of both free-skin grafts and skin flaps showed the same trend depending on time of irradiation after operation. The grafts or flaps irradiated in the hypervascular stage showed severe reactions to irradiation, whereas those irradiated in the hypovascular stage showed milder reactions in gross and microangiographic observation. Vascular damage should be given primary consideration when deciding the proper time for irradiation after reconstructive surgery. In general, free-skin grafts showed more severe reactions than skin flaps, especially in the grafts irradiated in the early stage after operation. The experimental results of this study cannot be readily transferred to a clinical setting, but they suggest that postoperative irradiation could be begun 3 to 4 weeks after operation with respect to graft or flap survival, and the results of the clinical cases almost coincide with these experimental results.     

Vascular island skin-flap tolerance to warm ischemia: an analysis by perfusion fluorometry

Fiberoptic perfusion fluorometry and assessment of ultimate viability were used to analyze the tolerance to warm ischemia of rat vascular island skin flaps. Both acute flaps and flaps raised 24 hours previously and then reraised were subjected to 0 (control), 6, 8, 10, and 12 hours of vascular pedicle clamping. Following clamp release, serial fluorometry documented the progressive delay in effective reflow resulting from extended periods of ischemia. Fluorometry, furthermore , suggested that flaps constructed 24 hours previously had an improved hemodynamic status with a significantly reduced period of poor reflow following clamp release. The improved hemodynamics were associated with increased viability, confirming the increased tolerance of 24-hour-old flaps to warm ischemia.     

Peripheral neovascularization of muscle and musculocutaneous flaps in the pig.

Late loss of free muscle flaps following surgical or accidental trauma to the dominant vascular pedicle has been reported. In this study, time-dependent ligation of the dominant vascular pedicle was undertaken in denervated latissimus dorsi musculocutaneous or muscle-only island flaps in the pig. Muscle flaps were covered with a skin graft, and silicon rubber sheets were inserted between the flaps and their bases to simulate a poorly vascularized bed. Hemodynamic and viability studies were then performed using intravenous fluorescein (skin viability), tetrazolium blue (muscle viability), and radiolabeled 15-micron microspheres (capillary blood flow). Blood flow did not change in acutely raised musculocutaneous flaps (n = 10) but was significantly elevated in acutely raised muscle-only flaps (n = 10), suggesting that the skin paddle may steal blood flow from the underlying muscle in musculocutaneous flaps. Peripheral neovascularization at 1 day to 8 weeks was assessed (n = 30). Viability increased during the first week of revascularization and was not different in musculocutaneous and muscle-only flaps. Revascularization of muscle-only flaps was enhanced compared with musculocutaneous flaps in the 2- to 8-week period.     

Effect of proteins depletion on the surviving length in experimental skin flaps.

Protein depletion appears to augment, rather than decrease, the length of a skin flap surviving in rats. We were able to document a lowering of serum viscosity in these animals. The augmentation of flap survival produced by protein depletion may even be greater than that produced by the delay phenomenon. A reduction in the total weight of flaps is present in the protein-depleted animals, and this may contribute to the augmentation of flap survival in them.