Critical ischemia times and survival patterns of experimental pig flaps

Previous work on critical ischemia time suggested (1) a greater susceptibility of myocutaneous flaps over skin flaps to the ischemia reperfusion injury and (2) that duration of ischemia may affect the survival area of a flap. Using a pig model, 55 animals were operated on and the critical ischemia times and survival patterns of the buttock skin (n = 85) and latissimus dorsi myocutaneous (n = 88) island flaps were determined after being submitted to 0, 2, 4, 6, 8, 10, 12, 14, and 16 hours of normothermic ischemia. The average critical ischemia times (CIT50) were determined to be 9 and 10 hours for the buttock skin and latissimus dorsi myocutaneous flaps, respectively. Percentage of total area surviving (%TAS) in those flaps which did survive was adversely affected by increases in the ischemic interval in both flap models. A statistically significant decrease in percentage of total area surviving was found after 6 and 8 hours of ischemia for the buttock skin and latissimus dorsi myocutaneous flaps, respectively.     

Secondary ischemia time in rodents: contrasting complete pedicle interruption with venous obstruction

The current study investigated the effect of secondary ischemic insults on ultimate flap survival. Rodent skin flaps subjected to 8 hours of secondary ischemia with total pedicle obstruction had 56 percent survival (7 of 12) compared with primary ischemic flaps of the same time, which all survived. At 10 hours of ischemia, only 42 percent of secondary ischemic flaps survived compared with 67 percent (8 of 12) of primary ischemic flaps. When the secondary ischemia was caused by venous obstruction, the results were even more striking. Ninety-two percent (11 of 12) of primary venous obstruction flaps survived 3 hours of ischemia and 75 percent (9 of 12) survived 5 hours of ischemia, while only 56 percent (7 of 12) and 8 percent (1 of 12) of flaps subjected to secondary venous obstruction survived at the same times, respectively. The explanation of these observations on the basis of tissue pathophysiologic changes will require further study. The results support the need for close monitoring of clinical flaps to ensure optimal survival.     

Preconditioning of latissimus dorsi muscle flaps with monophosphoryl lipid a

The use of dynamic myoplasty to restore function to failing organs is an exciting new application of skeletal muscle flaps. A complication of large flap elevation that can compromise flap function is ischemia-induced necrosis; one approach to minimizing this is to pretreat tissues with ischemic preconditioning. The purpose of this study was to determine whether systemic administration of monophosphoryl lipid A, a drug known to mimic late-phase ischemic preconditioning in the heart, could reduce ischemia-induced necrosis in latissimus dorsi muscle flaps. Forty latissimus dorsi muscle flaps from 20 Sprague-Dawley rats were allocated into four groups. In group I (n = 10), flaps were not preconditioned and served as controls. In group II (n = 10), flaps received ischemic preconditioning with two 30-minute periods of ischemia interspersed by 10 minutes of reperfusion. In group III (n = 10), rats received an intravenous bolus of approximately 0.3 ml of monophosphoryl lipid A vehicle only. In group IV (n = 10), rats received an intravenous bolus of 450 microg/kg of monophosphoryl lipid A and vehicle. Twenty-four hours after treatment, all latissimus dorsi muscle flaps were elevated on a single neurovascular pedicle and subjected to 4 hours of ischemia. After 72 hours of reperfusion, latissimus dorsi muscles were harvested, weighed, stained with nitroblue tetrazolium, and assessed for percent necrosis using digitized images of muscle sections and computerized planimetry. The percent necrosis in ischemic preconditioning-treated flaps (group II) was significantly reduced by 57 percent (p < 0.05) compared with control flaps (group I). The percent necrosis in flaps treated with monophosphoryl lipid A (group IV) was significantly reduced by 58 percent (p < 0.05) compared with vehicle-control flaps (group III). There was no difference in mean percent necrosis between ischemic preconditioning (group II) and monophosphoryl lipid A-treated (group IV) flaps or between ischemic preconditioning-control (group I) and monophosphoryl lipid A vehicle-control (group III) flaps. Intravenous administration of systemic monophosphoryl lipid A mimics the late-phase protective effect of ischemic preconditioning in the authors' rat latissimus dorsi muscle flap model.     

Neutrophil localization following reperfusion of ischemic skin flaps.

A swine model of island latissimus dorsi myocutaneous and buttock cutaneous flaps was used to examine neutrophil localization and flap survival after 6 hours of global ischemia followed by 24 hours of reperfusion. Radioactivity from autotransfused neutrophils labeled with indium-111 enabled their localization. Radioactivity in ischemic latissimus dorsi flaps was increased by 101 +/- 30 percent over contralateral control latissimus dorsi flaps (n = 6, p = 0.01). Radioactivity in ischemic buttock flaps was increased by 142 +/- 40 percent over contralateral control buttock flaps (n = 6, p = 0.008). Despite increased neutrophil localization to ischemic flaps, the magnitude of tissue radioactivity failed to provide sufficient information to predict ischemic injury as measured by flap survival and tissue water content.     

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.     

Vascular endothelial growth factor expression in pig latissimus dorsi myocutaneous flaps after ischemia reperfusion injury

Exogenous administration of vascular endothelial growth factor (VEGF) improves long-term viability of myocutaneous flaps. However, endogenous expression of this substance in flaps following ischemia-reperfusion injury has not been reported previously. Endogenous production of VEGF was measured in myocutaneous pig latissimus dorsi flaps after ischemia-reperfusion injury. Latissimus dorsi myocutaneous flaps (15 x 10 cm) were simultaneously elevated bilaterally in six Yorkshire-type male pigs (25 kg). Before elevation, three flap zones (5 x 10 cm) were marked according to their distance from the vascular pedicle. After isolation of the vascular pedicle, ischemia-reperfusion injury was induced in one flap by occlusion of the thoracodorsal artery and vein for 4 hours, followed by 2 hours of reperfusion. The contralateral flap served as a control. Perfusion in each zone was monitored by laser Doppler flowmetry at baseline, during ischemia, and during reperfusion. At the end of the protocol, skin and muscle biopsies of each flap zone and adjacent tissues were obtained for later determination of VEGF protein levels. VEGF concentrations were quantified using the Quantikine human VEGF immunoassay. Skin perfusion was similar among all flap zones before surgery. Flow fell in all flaps immediately after flap elevation. After 4 hours of ischemia, blood flow in the ischemic flaps was significantly decreased (p < 0.05) compared with nonischemic control flaps. After 2 hours of reperfusion, flow in ischemic flap skin recovered to levels similar to those in control flaps. VEGF protein concentrations in muscle tissue exceeded concentrations in skin and decreased from zones 2 to 3 in control and ischemic flaps. No significant differences in VEGF concentrations between ischemic and control muscle zones were observed. However, the concentration of VEGF in all muscle zones was significantly higher (p < 0.05) than muscle adjacent to the flap. Concentrations in skin zones 1 and 2 were significantly higher (p < 0.05) in ischemic flaps than in control flaps, but levels in zone 3 (most ischemic flaps) showed no significant difference.     

Vascular delay and administration of basic fibroblast growth factor augment latissimus dorsi muscle flap perfusion and function

Ischemia of the distal latissimus dorsi muscle flap occurs when the entire muscle is acutely elevated. Although this level of ischemia may not be critical if the muscle is to be used as a conventional muscle flap, the ischemia causes decreased distal muscle function if it is used for dynamic muscle flap transfer. This experiment was designed to determine whether or not the administration of exogenous basic fibroblast growth factor (bFGF), combined with a sublethal ischemic insult (i.e., vascular delay), would further augment muscle perfusion and function. Both latissimus dorsi muscles of nine canines were subjected to a bipedicle vascular delay procedure immediately followed by thoracodorsal intraarterial injection of 100 microg of bFGF on one side and by intraarterial injection of vehicle on the other. Ten days later, both latissimus dorsi muscles were raised as thoracodorsally based island flaps, with perfusion determined by laser-Doppler fluximetry. The muscles were wrapped around silicone chambers, simulating cardiomyoplasty, and stimulating electrodes were placed around each thoracodorsal nerve. The muscles were then subjected to an experimental protocol to determine muscle contractile function. At the end of the experiment, latissimus dorsi muscle biopsies were obtained for measurement of bFGF expression. The results demonstrated that the administration of 100 microg of bFGF immediately after the vascular delay procedure increases expression of native bFGF. In the distal and middle muscle segments, it also significantly increased muscle perfusion by approximately 20 percent and fatigue resistance by approximately 300 percent. The administration of growth factors may serve as an important adjuvant to surgical procedures using dynamic muscle flap transfers.     

Altered neutrophil function following reperfusion of an ischemic myocutaneous flap.

The neutrophil has been implicated as a source of oxygen free radicals provoking the reperfusion injury in various ischemic organs. This provided the motivation to explore the pathophysiologic role of the neutrophil in a swine model of postischemic latissimus dorsi myocutaneous flaps. Neutrophil function, neutrophil sequestration, and the anatomic distribution of muscle injury were estimated following a 6- to 8-hour global ischemic insult. Neutrophil function as measured by phorbol myristate acetate-stimulated superoxide production was found to be enhanced on reperfusion of ischemic flaps (n = 17). Neutrophil sequestration estimated from the arterial-venous difference of flap blood (n = 12) demonstrated that postischemic flaps more avidly sequester neutrophils than nonischemic flaps. The anatomic distribution of muscle injury (n = 7) was predominantly localized to the proximal portion of the ischemic flap. The enhanced functional response exhibited by neutrophils reperfusing an ischemic myocutaneous flap supports an active neutrophil role in the mediation of reperfusion injury.     

Differing flow patterns between ischemically challenged flap skin and flap skeletal muscle: implications for salvage regimens.

In this study, the authors tested the hypothesis that there is a significant difference in spatial patterns of reflow in skin as opposed to skeletal muscle after an ischemic insult. The authors believe that this pathophysiologic difference between the two flap types has significant implications for flap salvage strategies. Bilateral buttock skin flaps (10 x 18 cm) and latissimus dorsi myocutaneous flaps (10 x 20 cm) were elevated in Landrace pigs (n = 7). Flaps on one side of the animal were randomly assigned to 6 hours of arterial occlusion, with the contralateral side acting as control. At 15 minutes, 1 hour, and 4 hours after reflow, radioactive microspheres (15 microm) were injected into the left ventricle. After 18 hours of reperfusion, skin and muscle viability were estimated by intravenous fluorescein and soaking in nitroblue tetrazolium, respectively. Flow rates in the skin with an ischemia-reperfusion injury were significantly reduced (30 to 53 percent), at all time intervals, compared with controls. The flow rate in the fluorescent skin with ischemia-reperfusion injury of the latissimus dorsi flaps (0.037 ml/min/g at 15 min) was greater than in that of the buttock flaps (0.018 ml/min/g). The muscle flaps with ischemia-reperfusion injury had significantly higher flow rates than control muscle flaps at all time intervals studied (at 1 hour, 0.32 ml/min/g compared with 0.16 ml/min/g, respectively). In flap skeletal muscle, an early hyperemic phase during reperfusion maintains a significant blood flow to all regions, including the area of the flap that is destined for necrosis. In flap skin, however, there is a marked decrease in flow rates. These differences have important implications for the intravascular delivery of therapeutic agents to the damaged portions of the flap. Transdermal drug delivery systems should be explored as an alternative to intravascular regimens for the salvage of flap skin with ischemia-reperfusion injury.     

The effect of thromboxane synthetase inhibition on tolerance of skin flaps to secondary ischemia caused by venous obstruction

The harmful effects of the no-reflow phenomenon on skin flaps were modified by using the thromboxane synthetase inhibitor UK-38,485. Sprague-Dawley rats (N = 134) were subjected to either 3 or 5 hours of secondary venous occlusion occurring 24 hours after a primary ischemic episode of 1 1/2 hours. Within each time period, rats received either saline or UK-38,485 at the primary ischemic episode and/or at the secondary ischemic episode. Flaps treated with UK-38,485 in relation to the period of secondary ischemia had a higher survival rate than control ischemic flaps (p less than 0.01). Those treated only at the end of the primary ischemic episode but prior to the secondary ischemic episode had improved survival rates, but these were not statistically significant. These effects may be explained by the lower thromboxane:prostacyclin ratios at the time of revascularization. The possible interrelationship of the prostanoids with free-radical mechanisms in the no-reflow phenomenon is also discussed.     

Protective effects of superoxide dismutase against ischemia-reperfusion injury: development and application of a transgenic animal model

Reperfusion of ischemic tissues can be associated with structural and functional injury, which is referred to as ischemia-reperfusion injury. Superoxide dismutase is an endogenous free radical scavenger that converts toxic oxygen derived free radicals to hydrogen peroxide. With the development of gene cloning technology, the potential of manipulating cells to overexpress endogenous proteins has been realized. Transgenic mice capable of overexpressing superoxide dismutase, and knockout mice in which the gene responsible for its production has been deleted, were used as a model to examine the protective effects of superoxide dismutase against ischemia-reperfusion injury. Epigastric island flaps were elevated in wild-type (control), transgenic superoxide dismutase 1, and knockout superoxide dismutase 1 mice and subjected to ischemic intervals of 0, 3, 6, 9, or 12 hours. Five animals were studied at each time point in each study group. Flap viability was assessed on postoperative day 7. Baseline wild-type flap survival was 100 percent after 3 hours of ischemia and subsequent reperfusion; survival decreased to 21 percent after 9 hours of ischemia. Transgenic mice had significantly higher flap survival than wild-type animals after 6 hours of ischemia and subsequent reperfusion (97.0 versus 85.2 percent) and after 9 hours of ischemia (82 versus 21 percent, p < 0.01). In knockout mice, there was complete flap necrosis after as little as 3 hours of ischemia. This study confirms the protective effects of superoxide dismutase against ischemia-reperfusion injury. In addition, its deficiency results in a dramatic susceptibility to ischemic injury.     

The effects of the nitric oxide donor SIN-1 on ischemia-reperfused cutaneous and myocutaneous flaps

Ischemia-reperfusion injury causes tissue damage that leads to a decrease in bioavailability of nitric oxide. The authors hypothesized that an exogenous supply of nitric oxide will have beneficial effects on survival of skin and skeletal muscle subjected to ischemia-reperfusion injury. By using the nitric oxide donor SIN-1 (3-morpholino-sydnonimine) the effects of direct intraarterial infusion of an exogenous source of nitric oxide in reperfused flaps was studied. Bilateral island buttock skin flaps and latissimus dorsi myocutaneous flaps were elevated in eight pigs, for a total of 32 flaps. Flaps were subjected to 6 hours of ischemia followed by 18 hours of reperfusion. Flaps on one side of each animal were randomized to be treated with the nitric oxide donor (treatment group). The contralateral side was treated with an equivalent volume of saline vehicle (infusion control) SIN-1, or saline was administered as a continuous direct intraarterial infusion at the onset of reperfusion and continued during the observation period. Outcomes measured were tissue neutrophil accumulation by using myeloperoxidase assay and tissue survival (intravenous fluorescein and nitroblue tetrazolium for skin and muscle, respectively). In both skin and myocutaneous flaps, SIN-1 treatment caused a significant improvement in survival and a decrease in neutrophil accumulation. Nitric oxide may play an important role in the pathophysiologic process of ischemia-induced reperfusion injury in skin and skeletal muscle. Nitric oxide donors may be a promising family of therapeutic agents for the prevention of ischemia-induced reperfusion injury in cutaneous and myocutaneous flaps.     

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.     

Presence and activity of nitric oxide synthase isoforms in ischemia-reperfusion-injured flaps

Nitric oxide is produced from the amino acid L-arginine by nitric oxide synthase, which has three known isoforms: (1) endothelial nitric oxide synthase and (2) brain nitric oxide synthase, both of which are constitutive nitric oxide synthase; and (3) inducible nitric oxide synthase. The authors' hypothesis is that after reperfusion injury, endothelial cell dysfunction leads to disruption of nitric oxide synthase-mediated nitric oxide production and that this may in part explain the deleterious effects of ischemia-reperfusion injury on tissue survival and blood reflow in flaps. An experiment was designed to study the effects of ischemia-reperfusion injury on the bioactivity of all three isoforms of nitric oxide synthase. Buttock skin flaps and latissimus dorsi myocutaneous flaps were elevated in eight pigs. Flaps on one side of the animal were randomized to receive 6 hours of arterial ischemia, whereas flaps on the other side served as controls. At 6 hours of ischemia and at 1, 4, and 18 hours after reflow, tissue biopsy specimens were obtained and were processed for both constitutive nitric oxide synthase and inducible nitric oxide synthase enzyme activity on the basis of the L-citrulline assay. In addition, specimens were processed for Western blot analysis of the three isoforms. The authors' results revealed three key findings: first, there was a statistically significant (p < 0.001) decrease in constitutive nitric oxide synthase activity of ischemia-reperfusion-injured flaps as compared with controls in both skin and muscle for all time intervals measured. Second, Western blot analyses of endothelial nitric oxide synthase and brain nitric oxide synthase showed a significant decrease in the signal intensity in ischemic and reperfused tissue as compared with controls. Third, the inducible nitric oxide synthase isoform's activity and protein remained undetectable in both tissue types for all time points measured. The authors' data demonstrated that following ischemia-reperfusion injury in the pig flap model there was a disruption of constitutive nitric oxide synthase expression and activity, which may lead to decreased nitric oxide production. The significant decrease in nitric oxide synthase activity found in the current study may partly explain the mechanism of tissue damage in flaps subjected to ischemia-reperfusion injury. Knowledge of the kinetics of nitric oxide synthase activity under conditions of ischemia-reperfusion injury has important implications for the choice and timing of delivery of therapeutic agents whose goal is to increase the bioavailability of nitric oxide in reperfused tissue.     

Ischemia-related changes in galanin expression in the dentate hilar region after transient forebrain ischemia in gerbils.

Although galanin (GAL) protects hippocampal neurons from ischemic damage, no study has examined ischemia-related changes in endogenous GAL in the hippocampal dentate gyrus. We investigated the chronological changes of GAL, well-known as the potassium channel opener, expression in the dentate gyrus at various times after 5 min of transient forebrain ischemia in gerbils. A few GAL-immunoreactive (IR) neurons were found in the polymorphic layer of the sham-operated group. Three hours after ischemia-reperfusion, the pattern of GAL immunoreactivity was similar to that of the sham-operated group and the number of GAL-IR neurons and immunoreactivity were highest 12 h after ischemic insult. At this time, GAL-IR neurons in the polymorphic layer showed strong GAL immunoreactivity. Thereafter, GAL-IR neurons and immunoreactivity significantly decreased in the dentate hilar region. Four days after ischemic insult, GAL-IR neurons were not detectable. In addition, the results of a Western blot study showed a pattern of GAL expression similar to the immunohistochemical changes. GAL protein content also was highest 12 h after ischemia. In conclusion, the increased expression of endogenous GAL in the dentate gyrus after ischemia is related to response to the ischemic damage.     

Early and late effects of ischemic preconditioning on microcirculation of skeletal muscle flaps

The present study was designed to investigate the early and late effects of ischemic preconditioning on muscle flap perfusion and reperfusion-induced skeletal muscle damage. Thirty-six Sprague-Dawley rats were divided into six experimental groups of six animals each. The cremaster muscle flap model and the intravital microscopy system were used to observe microcirculatory changes associated with ischemia-reperfusion injury and ischemic preconditioning. In groups 1, 2, and 3, microcirculatory measurements were taken on the same day; however, in groups 4, 5, and 6, measurements were taken a day after surgery. Group 1 served as a control. The cremaster muscle was prepared as a tube flap, subjected to an hour of perfusion without ischemia. In group 2 (ischemic preconditioning + ischemia group), the cremaster muscle tube flap was subjected to 30 minutes of ischemia and 30 minutes of reperfusion, followed by 4 hours of total ischemia. In group 3 (ischemia alone), the flap was submitted to 4 hours of ischemia alone. In group 4 (control), the cremaster muscle flaps were dissected out, preserved in the subcutaneous tunnel, and submitted to 24 hours of perfusion only. In group 5 (ischemic preconditioning + 24 hours of perfusion + 4 hours of ischemia), the ischemic preconditioning protocol was followed by 24 hours of perfusion and 4 hours of ischemia. In group 6 (24 hours of perfusion + ischemia), the same protocol was used as in group 5 without ischemic preconditioning. Functional capillary perfusion, and the diameters of the arterioles of the first, second, and third order were significantly increased in the ischemic preconditioning group during the early period, but not after 24 hours of perfusion. No differences in the red blood cell velocities of arterioles of the first, second, or third order were found in either the early-effect or late-effect groups. The numbers of rolling, adhering, and transmigrating leukocytes, however, were significantly lower in the ischemic preconditioning group at both early and late follow-up. Ischemic preconditioning of the skeletal muscle flap has both an early and a late protective effect against reperfusion injury. Ischemic preconditioning at the early interval significantly improves muscle flow hemodynamics of the flap and attenuates leukocyte-mediated reperfusion injury. After 24 hours of reperfusion, however, ischemic preconditioning failed to improve the flow hemodynamics of the flap, yet it still protected the skeletal muscle flap from leukocyte-mediated reperfusion injury.     

Improved perfusion after subcritical ischemia in muscle flaps treated with vascular endothelial growth factor

Vascular endothelial growth factor (VEGF), a potent endothelial mitogen, is secreted in ischemic tissue and plays a pivotal role in angiogenesis. We studied whether VEGF administered to a rat muscle flap at the time of ischemia induction would increase microcirculatory flow to the flap. The cremaster muscle flap was isolated on its neurovascular pedicle. Ischemia was induced by clamping the vascular pedicle, and 0.2 ml of either VEGF (0.1 microg) or vehicle (phosphate-buffered saline) was immediately infused into the muscle. After 4 or 6 hours, the clamps were released, and the cremaster was placed in a pocket in the medial thigh for 24 hours. The muscle was then dissected, and microcirculatory measurements were made under intravital microscopy. Six animals were used in each of the four groups. All flaps exposed to 6 hours of ischemia, the duration considered to be critical ischemia, had no significant microcirculatory flow, regardless of treatment with VEGF. In the 4-hour ischemia group, or subcritical ischemia group, red blood cell velocity in arterioles was 14 mm/sec in muscles treated with VEGF and 9 mm/sec in controls (p = 0.02), and capillary flow was 7 per high-power field in muscles treated with VEGF versus 2 per high-power field in controls (p = 0.0005). Thus, VEGF did not alter microcirculatory flow in a muscle flap exposed to critical ischemia, but it did enhance flow to a flap exposed to subcritical ischemia.     

Beneficial effects of ibuprofen on experimental microvascular free flaps: pharmacologic alteration of the no-reflow phenomenon

Pharmacologic alteration of the no-reflow phenomenon was determined based on increased tolerance to ischemia in ibuprofen-treated free flaps. Sprague-Dawley rats (N = 60) were divided into control (lactated Ringer's) and treated (ibuprofen) groups and subdivided into six groups of ischemia: 1 hour, 6 hours, 8 hours, 10 hours, 12 hours, and 14 hours of ischemia. Fluorescein uptake was measured after 10, 30, and 60 minutes following microrevascularization. Dye elimination studies were done for each ischemia group that demonstrated good fluorescein uptake. All free flaps in the 1-, 6-, and 8-hour groups survived. The ibuprofen-treated 10- and 12-hour flaps all survived, whereas the 10-hour control and 14-hour ibuprofen-treated free flaps failed to survive. Despite high fluorescein uptake, the 14-hour ibuprofen-treated flaps did not eliminate the fluorescein, whereas all surviving free flaps adequately eliminated the fluorescein. Failure to eliminate dye despite adequate uptake suggested a deranged microcirculation with increasing ischemia time. By inhibiting cyclo-oxygenase, nonsteroidal anti-inflammatory agents such as ibuprofen may block the untoward effects mediated by thromboxane A2, such as vasoconstriction, microvasculature thrombus formation, and intravascular sludging. These effects are theorized in part to be responsible for the failure of a free flap to survive despite revascularization.     

The role of perfusion washout in limb revascularization procedures

Amputated rat hindlimbs were subjected to either normothermic (26 degrees C) or hypothermic (4 degrees C) ischemia. Experimental limbs had their microcirculation washed out (either before or after the ischemic insult) with a physiologic acellular plasma substitute previously reported to enhance flap survival following extended periods of warm ischemia. Control limbs were not washed out; i.e., stagnant blood remained in these limbs. Following the ischemic interval, amputated limbs were replanted. Monastral blue B, a colloidal pigment capable of labeling leaky blood vessels, was administered systemically to all rats just prior to vascular declamping. Limb biopsies of skin and muscle were harvested 30 minutes following revascularization in order to assess Monastral labeling and, therefore, the functional integrity of the microcirculation. Results confirm that stagnant blood under conditions of warm ischemia is detrimental to the functionality of the microcirculation in both skin (p less than 0.03) and muscle (p less than 0.007). Accordingly, perfusion washout, when performed prior to the ischemic period, enhances limb survival following 6 hours of warm ischemia (p less than 0.01). Hypothermia protects against the detrimental effects of stagnant blood; perfusion offers no benefit if hypothermic conditions prevail. Physiologic mechanisms responsible for these findings are discussed.     

Delayed microcirculatory hyperpermeability following perfusion washout

Recent experimental data have demonstrated improved flap survival following perfusion washout with a synthetic, chemically defined, mammalian plasma. In an effort to define the physiology responsible for the efficacy of perfusion, the method of "labeling" hyperpermeable blood vessels with Monastral blue B in rat epigastric vascular island flaps was utilized. Results confirmed that capillary and venular hyperpermeability is an early and progressive pathophysiologic event in ischemic flap tissue and one which is reversible prior to a critical ischemic period. Perfusion washout with a physiologic, acellular plasma substitute delays the onset of vascular hyperpermeability. This may be a mechanism responsible for improving tissue survival following extended periods of warm ischemia (12 hours). It is implied that stagnant blood and products of hemolysis in the microcirculation may be detrimental to the functional and anatomic integrity of the endothelial wall.