Epigastric Island Flap Research Articles

Platelet-derived growth factor-AA-mediated functional angiogenesis in the rat epigastric island flap after genetic modification of fibroblasts is ischemia dependent

Background. The aim of this study was to induce therapeutic angiogenesis in ischemically challenged flap tissue by means of gene transfer. Methods. Isogenic rat fibroblasts were retrovirally transfected to produce platelet-derived growth factor (PDGF)-AA. Stable gene expression was monitored by PDGF-AA enzyme-linked immunosorbent assay. Eighty animals were divided into 2 groups (1 and 2), each with 4 subgroups. The angiogenic target was a 7 × 7-cm epigastric island flap used as a necrosis model. Group 1 received flap treatment 1 week before flap elevation: 107 genetically modified fibroblasts, expressing PDGF-AA (genetically modified fibroblasts) plus 1 mL of Dulbecco's modified Eagle's medium (DMEM) (1A), 107 nonmodified fibroblasts (NMFB) plus 1 mL of DMEM (1B), 1 mL of DMEM (1C), and 1 mL of sodium chloride 0.9% (1D). All substances were injected at evenly distributed spots into the panniculus carnosus of the entire flap. Group 2 had the same flap treatment at the day of flap elevation. All flaps were sutured back. Seven days later, the flaps were harvested and examined both clinically, histologically, and immunohistochemically. Results. In vitro, the GMFB produced up to 117.9 ± 57.2 ng of PDGF-AA/mL medium during a 4-day period, compared with 0.7 ± 0.6 ng of PDGF-AA/mL medium produced by NMFB in the same time period. In vivo production of PDGF-AA in flaps amounted to 1.3 ± 0.7 ng of PDGF-AA/1 μL flap tissue for group 1A and 1.7 ± 1.1 ng of PDGF-AA/1 μL flap tissue for group 2A seven days after cell transplantation. Fibroblasts persisted in all flaps from groups 1A, 1B, 2A, and 2B without major inflammatory reaction. Clinically, group 2A developed significantly less flap necrosis compared with all other groups, including group 1A. Accordingly, only group 2A gave significant histologic and immunhistochemical evidence for enhanced angiogenesis within the flap tissue. Conclusions. After retroviral gene transfer, isogenic rat fibroblasts produce high amounts of PDGF-AA in vitro. In vivo, PDGF-AA can be detected in flaps receiving genetically modified fibroblasts, which suggests survival of the implanted fibroblasts in this model. PDGF-AA produced by GMFB can induce flap angiogenesis only under ischemic conditions in this model. Transplantation of PDGF-AA-overexpressing fibroblasts results in higher flap survival in this model. (Surgery 2002;131:393-400.)

Protective effect of fucoidin (a neutrophil rolling inhibitor) on ischemia reperfusion injury: experimental study in rat epigastric island flaps.

The objective of this study was to examine whether a decrease in neutrophil-mediated tissue injury using Fucoidin, a nontoxic neutrophil rolling inhibitor, would improve flap survival in an island flap model after ischemia-reperfusion. Myeloperoxidase activity (an indirect index of tissue neutrophil count) and malondialdehyde (an indicator of lipid peroxidation), the degree of neutrophil infiltration by direct counting, and macroscopic flap survival were assessed in the flap after arterial ischemia-reperfusion. Epigastric island skin flaps were elevated in 56 rats. The first group of 21 rats was subjected to 6 hours of arterial ischemia. The second group of 21 rats was subjected to 10 hours of arterial ischemia, and the rest of the rats were used as nonischemic controls (sham flaps). For inhibiting neutrophil rolling, a nontoxic polysaccharide agent-Fucoidin-was used. Each ischemic group was divided further into three subgroups: Subgroup I (control rats) received saline, subgroup II received 10 mg per kilogram Fucoidin, and subgroup III received 25 mg per kilogram Fucoidin before reperfusion. The results were evaluated as tissue neutrophil counts, tissue malondialdehyde content, tissue myeloperoxidase activity, and flap survival. Neutrophil counts and tissue myeloperoxidase activity were decreased significantly (p <0.001) in subgroup III, but lipid peroxidation by means of tissue malondialdehyde content was not affected by Fucoidin administration. The authors conclude that administration of Fucoidin before reperfusion can limit tissue injury apparently by inhibiting neutrophil rolling in a dose-dependent manner.

The effect of dipyrone on survival of skin flaps.

The effect of dipyrone on the skin flap survival was studied in a model of an epigastric island flap with a random extension on the opposite side in 23 rats. Dipyrone was given intraperitoneally one hour before the skin flap was raised. At the end of the operation the depth of penetration of the fluorescein dye in the skin flap was assessed visually from photographic records and the flap survival area was measured by a grid method on the seventh postoperative day. There was a significant reduction in the amount of ischaemic necrosis of the skin flap after a single dose of dipyrone 50 mg/kg (p < 0.001). These data suggest that dipyrone is a useful agent in the prevention and treatment of ischaemia and necrosis of the skin flap.

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.

Effect of surgical denervation on the viability of inferior epigastric neurovenous flaps in the rat.

The purpose of this study was to examine how the inferior epigastric neurovenous flap in the rat reacts to surgical denervation. The survival of a denervated flap was compared with that of an innervated flap. A 2 x 2-cm flap was raised in 30 female Wistar rats assigned randomly to six groups of 10 rats each: group 1, innervated neurovenous flap; group 2, denervated neurovenous flap, acute model; group 3, denervated neurovenous flap, chronic model; group 4, innervated inferior epigastric island flap; group 5, denervated inferior epigastric island flap, acute model; and group 6, control, composite graft. Acute denervation produced a significant decrease in the survival of the inferior epigastric neurovenous flap (p < 0.05). The surviving area of the innervated flaps decreased from 94+/-14% (mean +/- standard deviation) to 16+/-34% by acute denervation. Chronic denervation was effective in decreasing flap necrosis in these flaps (survival, 99+/-5%). There were no differences between the average viable area of the standard inferior epigastric flap in the denervated and innervated groups.

Genetically modified fibroblasts induce angiogenesis in the 3×6 cm rat epigastric island flap

The hypothesis as to whether gene therapy may induce functional angiogenesis in the rat superficial epigastric island flap would allow earlier pedicle division, was tested and the results are presented. Autologous rat fibroblasts were grown, harvested, cultured and retrovirally transfected to produce platelet-derived growth factor (PDGF)-AA, an angiogenetically active protein. Stable gene expression was monitored by PDGF-AA enzyme-linked immunosorbent assay (ELISA). One hundred eighty animals were divided into three groups (I-III) and a bilateral flap created in each animal. In all experiments, the right-sided flap was subjected to the experimental treatment and the left-sided flap served as control (1 ml saline 0.9%). During flap elevation, group 1 received 5×106 genetically modified fibroblasts (GMFB ) plus 1 ml DMEM as medium. Group 2 was treated with 5×106 non-modified fibroblasts (NMFB ) plus 1 ml medium; and group 3 received 1 ml medium alone. The flaps were sutured back and the vascular pedicle was bilaterally ligated and divided in each batch of 10 animals during the following 6 days. Seven days later, the flaps were harvested, the amount of necrosis measured and histologically examined. The GMFB produced up to 560 times more PDGF-AA than the NMFB, measured by ELISA. Clinically, the GMFB-treated flaps tolerated surgical division of the vascular pedicle significantly earlier than groups 2 and 3. Histologically, fibroblasts persisted in all flaps of groups 1 and 2 without major inflammatory reaction. In all GMFB-treated massive angiogenesis could be demonstrated. By means of retroviral gene transfer autologous rat fibroblasts can be genetically modified for stable expression of the PDGF-A gene to produce high amounts of PDGF-AA, which is angiogenetically active. After injection into the panniculus carnosus, these cells induce functional angiogenesis to permit earlier division of the vascular pedicle in this flap model.

Physiological roles of endothelium-derived nitric oxide in the epigastric island flaps of rabbits.

Nitric oxide (NO), identified as the mediator of endothelium-dependent relaxation of vascular smooth muscle, is known to cause a number of inflammatory conditions, especially in ischemia/reperfusion injury. This experimental study, using a rabbit epigastric island flap, was designed to investigate whether skin flap ischemia followed by reperfusion-influenced serum NO and c-GMP concentrations in the flap. In addition, we also investigated the premedicated effects of the NO synthase inhibitor and heparin on serum NO and c-GMP concentrations in skin flap ischemia/reperfusion. Serum NO concentration after 15, 30, 45, and 60 minutes of ischemia followed by reperfusion significantly increased compared with that in nonischemic control and elevated flaps. On the contrary, serum NO concentration was suppressed in L-NAME or aminoguanidine pretreated animals with ischemic group. Administration of heparin increased the serum NO concentration in elevated flaps, but suppressed it in ischemic flaps followed by reperfusion. The changes in serum c-GMP and NO concentrations were related in all of the experimental groups. These results suggest that NO may be derived from vascular endothelial cells and dilate peripheral vessels in compensation for ischemia.

The effect of washout perfusion with beraprost sodium on the no-reflow phenomenon in the rat island flap

Beraprost sodium is a chemically stable PGI2 analog which has antiplatelet and vasodilating properties, its ability to improve survival after prolonged ischemia was tested on the rat epigastric island flap. Forty Sprague-Dawley rats were divided into control (Ringer's lactated) and treated (beraprost sodium) groups and subdivided into two groups of total ischemia (both artery and vein were occluded): 11 and 15 h. After the ischemic period the flaps were slowly perfused with 4 ml of beraprost sodium solution or R/L solution. After seven days the survival areas were documented: Goup 1 (control — 11 h of ischemia): 9.9%; Group 2 (beraprost sodium — 11 h of ischemia): 85.9%; Group 3 (control — 15 h of ischemia): 0%; Group 4 (beraprost sodium — 15 h of ischemia): 48.2%. Using the student's t-test the result demonstrated that beraprost sodium improved flap survival in both the 11 and 15 h of ischemia groups (p<0.005).

The influence of skin flap ischemia on serum nitric oxide concentrations.

Nitric oxide (NO), identified as a mediator of endothelium-dependent relaxation of vascular smooth muscle, is known to cause a number of inflammatory diseases, especially ischemia-reperfusion injury. This experimental study, using a rabbit epigastric island flap, was designed to investigate whether skin flap ischemia followed by reperfusion influences serum NO concentrations. In addition, the author investigated the effects of NO synthase inhibitors and heparin on skin flap ischemia. Serum NO concentrations after 15, 30, 45, and 60 minutes of ischemia followed by reperfusion were significantly increased compared with non-ischemic controls and elevated flaps. On the other hand, serum NO concentrations were suppressed in nitro-amino-methyl-L-arginine- and aminoguanidine-treated animals. Furthermore, administration of heparin increased serum NO concentrations in controls and animals with elevated flaps, but decreased serum NO concentrations in ischemic flaps with subsequent reperfusion. These results suggest that NO is one of the factors responsible for ischemia-reperfusion injury and that NO synthase inhibitors and heparin may protect against such injury.

Assessment of acute microcirculatory changes by color Doppler sonography

We studied the efficacy of color Doppler sonography in the transcutaneous assessment of acute changes in microvascular flow. Arterial and venous occlusion studies were performed in rabbits after simple isolation of the common femoral artery and vein and after raising an epigastric island flap. Vessel diameters were measured through an operating microscope and compared to color Doppler determinations of the diameters. Vessel patency and blood flow in the major vessels were monitored before and after occlusion. The correlation of vessel diameters measured under the operating microscope and with color Doppler sonography was .91 for the arteries and .39 for the veins. A strong linear correlation existed between Doppler identification of arterial occlusion, but not of venous occlusion. This study demonstrated that color Doppler sonography can be used transcutaneously to reliably assess patency and blood flow in the small vessels. Time-dependent, reproducible patterns in the spectral waveform analysis were indicative of the onset of arterial or venous occlusion.

Vascular Freezing—A New Method for Immediate and Permanent Vasospasm Relief

Although much work has been done on the etiology and prevention of vasospasm, a spasmolytic agent capable of firmly protecting against or reversing vasospasm has not yet been found. In this paper, we describe a new physical method of treating vasospasm. It consists of directly freezing the arterial walls. Experiments were performed on the epigastric artery of 40 female Sprague-Dawley rats. A vasospasm was created by mechanical vessel manipulation. The vasospasm was treated by freezing the vessel walls with a dermatologic hand-held liquid nitrogen spray. Vascular spasm and patency controls were made immediately after thawing and 30 minutes and 3, 10, and 120 days after freezing. Epigastric island flaps were harvested based on the frozen artery at 3, 10, and 120 days after the freezing procedure. Histologic studies also were effectuated 3, 10, and 120 days after the freezing procedure. Relief from vasospasm was instantaneous and permanent. After freezing, almost all the cells in the media and endothelium died and the adrenergic fibers degenerated. This was followed by a phagocytic debridement, complete regeneration of the endothelium, and a limited regeneration of the muscle fibers in the media. The adrenergic innervation was recovered, and no alterations in the adrenergic fibers distal to the lesion were noted at any time. None of the arteries examined underwent thrombosis, and all the flaps harvested survived very well, indicating that the vascular bed distal to the site of the freezing was normal. This vascular freezing technique may well have clinical applications for microsurgical transfers and for the prophylactic treatment of vasospasm disease. However, its indications still require clarification.

Flap tolerance to ischaemia in streptozotocin-induced diabetes mellitus

The effects of diabetes mellitus (DM) on skin flap tolerance to 3 h of secondary venous ischaemia were evaluated. Epigastric island flaps were elevated 3, 6 and 12 weeks after induction of DM in rats. In the non-diabetic control groups, the flap survival was 85% in the 3-week group, 72% in the 6-week, and 78% in the 12-week. In untreated DM groups, the flap survival significantly decreased to 40% in the 3-week group, 25% in the 6-week, and 17% in the 12-week (P < 0.05 in all groups). Flap survival in the DM/insulin group decreased to 31% in the 3-week group. Effects of insulin therapy, however, were observed in later stages of DM: 71% and 62% survival in the 6- and 12-week group, respectively. Significant linear correlations between enzymatic responses and the flap survival were found. The results suggest that DM is detrimental to flap tolerance and is associated with the lack of metabolic responses to secondary ischaemia.

Effect of skin flap ischemia on plasma endothelin-1 levels.

A number of studies have been done relating to vasospasm. Vasospasm within the microvasculature of a flap can be one of the causes of ischemia and nonviability. Endothelin-1 (ET-1), a 21-amino acid polypeptide isolated from vascular endothelium culture media, is reported to be one of the most potent vasoconstrictors known. This experimental study, using a rabbit epigastric island flap, was designed to investigate whether skin flap ischemia influenced plasma ET-1 levels. After the ischemic insult, blood was drawn from the venous effluent of the flaps. Plasma ET-1 levels after 6 hours of ischemia were significantly increased compared with nonischemic controls; they were 29 pM, i.e., almost enough to induce vasoconstriction of arterioles. These results suggest that ET-1 is one of the factors responsible for partial necrosis of the skin flap, which contributes to the genesis of the no-reflow phenomenon.

The biochemical basis of secondary ischemia

In this study rat epigastric island flaps were used as a model to investigate selected tissue biochemical changes occurring during secondary ischemia. It was hypothesized that free radical damage, depletion of free radical scavengers, depletion of ATP, and increased edema might explain differences in flap survival between partial (venous obstruction) and total (arteriovenous obstruction) ischemia and decreased flap survival with increasing ischemia time. Flaps were given 2 hr of primary ischemia, 8 hr of normal perfusion, then secondary ischemia of 0, 2, 4, 8, or 12 hr with either arteriovenous obstruction or venous obstruction. Biochemical analysis of the skin was performed after 0, 24, or 96 hr reperfusion. Only minor differences were found between arteriovenous and venous ischemia for any of five biochemical parameters, despite a previous finding that venous ischemic flaps are more susceptible to necrosis. Levels of xanthine oxidase and malonyldialdehyde (both indices of free radical generation) increased with ischemia time. Levels of superoxide dismutase (a free radical scavenger) correspondingly decreased. Tissue levels of ATP decreased after ischemia and recovered to normal for shorter but not for longer ischemia times after 96 hr of reperfusion in parallel with flap survival. Edema increased immediately after the ischemic insult but decreased once the tissue became necrotic. These results imply roles for free radicals, ATP, and edema in secondary ischemia, but do not distinguish between arteriovenous and venous secondary ischemia.

Leeches: controlled trial in venous compromised rat epigastric flaps

Leeches were studied for their efficacy to improve survival of venous compromised rat skin flaps. In 22 rats, bilateral epigastric island flaps were created and subjected to 6 h of venous occlusion. One flap in each animal was randomised to leech treatment, while the contralateral flap served as its own control. Flap survival, leech feeding time, weight gained by the leech and bleeding time from leech bites were measured. The area of flap survival was significantly increased in leech treated flaps compared to contralateral controls (n = 22 pairs, p = 0.03; Wilcoxon signed rank test). Weight gained in the feeding leech averaged 1.3 ± 0.2 g (n = 18). Leech feeding time was 107 ± 13 min (n = 18). Bleeding time from each leech bite averaged 79 ± 12 min (n = 18). Hence, the extent of flap necrosis resulting from venous impairment can be partly diminished by leech treatment until definitive surgical venous revascularisation.

EFFECT OF A THROMBOXANE SYNTHETASE INHIBITOR UK‐38, 485 ON THE TOLERANCE OF SKIN FLAPS OF PRIMARY ISCHAEMIA

The harmful effects of ischaemia or skin flaps were modified using the thromboxane synthetase inhibitor UK-38,485. The epigastric island flaps of Sprague-Dawley rats (n = 288) were subjected to 10, 12 or 14 h of total pedicle occlusion, or 3, 5 or 7 h of venous occlusion of the sole vascular pedicle. Within each time period, rats received intravenous doses of either physiological saline (controls) or UK-38,485 at the beginning or end of the ischaemic episode. Flaps treated with UK-38,485 overall had a higher survival rate than control ischaemic flaps (P less than 0.001). This applied both to total (arterial) ischaemia (P less than 0.001) and partial (venous) ischaemia (P less than 0.01). There was no significant difference between treatment given at the beginning or at the end of the ischaemic episode. These results may be explained by reduced platelet aggregation and thrombosis in the microvasculature due to the lower thromboxane/prostacyclin ratios for treated flaps. The possible inter-relationship of the prostanoids with free radical mechanisms in the no-reflow phenomenon is also discussed.

Thoracodorsal and caudal superficial epigastric axial pattern skin flaps in cats.

Nonselective angiography, selective angiography, and gross dissection of 15 cadavers were performed to delineate direct cutaneous arteries in the cat. The omocervical, deep circumflex iliac, thoracodorsal, and caudal superficial epigastric arteries were identified by nonselective angiography. Selective angiography and gross dissection allowed assessment of the origin and vascular territories of the thoracodorsal and caudal superficial epigastric arteries. Orthotopic and heterotopic transfers of thoracodorsal and caudal superficial epigastric island flaps were performed on eight cats. All flaps were successful although areas of necrosis at the caudodistal tips were evident in most of the thoracodorsal flaps. The rotated thoracodorsal flaps extended to the carpi. Caudal superficial epigastric flaps enabled coverage to the metatarsus. Seroma formation and partial dehiscence were minor complications.

The effect of time of vascular island skin flap elevation on tolerance to warm ischemia.

The effect of time of vascular island skin flap elevation on tolerance to a subsequent 12-hour period of warm ischemia was studied. Sixty rats were separated into five equal groups; the rat epigastric island flap was used as the model. In group 1 flaps were elevated and immediately subjected to 12 hours of complete ischemia by application of microvascular clamps to both artery and vein of the pedicle. In group 2 the flap was elevated, and after 12 hours of recovery ischemia was induced for 12 hours. Groups 3 to 5 were similar to group 2 except that the time interval between initial elevation and subsequent ischemia varied: 24 hours for group 3; 72 hours for group 4; 144 hours for group 5. Necrosis was evaluated on postoperative day 7. Those flaps elevated 24 hours before ischemic insult (group 3) had significantly better survival than all other groups (p less than 0.025 at least). There were no significant differences between the other groups. Flap elevation 24 hours before a complete ischemic episode significantly increased tolerance to ischemia. Elevating a flap earlier or later than 24 hours did not have any significant benefit.

Effect of the vasodilator peptides calcitonin gene-related peptide and atriopeptin on rabbit microcvascular blood flow. Preliminary communication

A neuropeptide known as calcitonin gene-related peptide (CGRP) has been shown to have vasodilatory properties in the rabbit epigastric island flap of almost equivalent potency to prostacyclin. The latter is currently regarded as the most potent vasodilator yet to be isolated. CGRP, with its longer-lasting effect in blood and its ability to overcome noradrenaline-induced vasospasm, may be promising for use during or after ischaemic flap transfer or replantation. Another known vasodilatory peptide, atriopeptin, was not as potent in the microvascular bed of the cutaneous flap as in major blood vessels.

Correlation of viability and laser Doppler flowmetry in ischemic flaps

Microvascular blood flow was measured by a laser Doppler flowmeter in rabbit epigastric island flaps made ischemic at 37°C for either 6.5 hr (Group 1; n = 13) or 17.5 hr (Group 2; n = 4). Contralateral flaps raised without ischemia were used as controls. The survival of ischemic flaps in these groups at 7 days was 69 and 0%, respectively. After 6.5 hr of ischemia, microvascular blood flow in the ischemic flap was approximately 20% below that in the control flap for the first 3–4 days postoperatively, presumably reflecting ischemic damage to parts of the cutaneous microcirculation; thereafter, flow was not significantly different from controls up to 7 days postoperatively. Failed 6.5-hr ischemic flaps had initial sluggish blood flow which slowed to zero usually within 4 days postoperatively. After 17.5 hr ischemia, negligible microvascular flow was detectable postoperatively. Failure in the microcirculation as detected by the flowmeter occurred well in advance of any noticeable physical changes in the flap. It is concluded that the percentage ischemic flap flow of control flap flow for each animal (at any selected time up to 24 hr after revascularization) reliably predicts the viability of the flap after 7 days.