Improved survival in free skin flap transfers in rats

We have demonstrated previously that oxygen-derived free radicals are important mediators of tissue injury in experimental island skin flaps that have been subjected to prolonged ischemia (vascular occlusion) followed by reperfusion. In this study the role of oxygen free radicals in ischemia/reperfusion injury has been investigated in free flap transfers. Groin skin flaps were harvested, stored at room temperature for 21 to 24 hours, and transplanted to the contralateral groin. These free flap transfers normally exhibit a high incidence of complete necrosis. Treatment before the onset of reperfusion with a single dose of superoxide dismutase (SOD), a scavenger of superoxide radicals, increased the survival rate of these skin flaps from 38% in the control group to 76% (p less than 0.025). Tissue levels of SOD were measured before ischemia, after ischemia but before reperfusion, and 30 minutes after reperfusion: untreated flap tissues, which were destined to undergo necrosis, exhibited a significant decrease in SOD activity after reperfusion, whereas SOD-treated flap tissues, destined to survive, demonstrated increased enzyme activity. High levels of tissue SOD activity thus appeared to be associated with improved flap survival. The results have significant clinical implications with regard to organ preservation and transplantation.     

The role of oxygen-free radicals in ischemic tissue injury in island skin flaps

The contribution of free radical-mediated reperfusion injury to the ischemic damage caused by total venous occlusion of island skin flaps was investigated in a standardized rat model. Control flaps subjected to 8 hours of total venous occlusion showed complete, full thickness necrosis when followed for 7 days following release of the vascular occlusion. Treatment with superoxide dismutase, a scavenger of superoxide radicals, prior to and immediately following the onset of reperfusion, significantly enhanced island flap survival from 0/11 (0%) to 8/15 (53%), p less than 0.005, and from 0/9 (0%) to 6/12 (50%), p less than 0.02, respectively. These findings are consistent with the hypothesis that oxygen free radicals generated at the time of reperfusion following a period of ischemia contribute significantly to the ultimate damage caused by ischemic injury. Such findings are consistent with similar reported observations on other tissues and suggest a means by which ischemic tissue injury might be therapeutically modified, even after the period of ischemia.     

Effect of superoxide dismutase for improvement of survival of ischemic reperfused island skin flap]

Using a rat model, we evaluated the effect of SOD on the survival of ischemic reperfused island skin flaps. In experiment 1, the oxygen free radical concentration in the flaps was measured by the technique of ESR. The results showed that the oxygen free radical concentration in ischemic reperfused flaps was significantly higher than in the corresponding control flaps (P less than 0.001). In experiment 2, the flaps were perfused with SOD (2000 U in 1 ml saline) before reperfusion after 8 hours of ischemia. Seven days after operation, the area of flap survived in the test group was significantly larger than in the control group (P less than 0.0005). The obtained data demonstrated that the generation of oxygen free radical increases with time during ischemia reperfusion in island skin flap and the role of oxygen free radical in tissue injury following ischemia and reperfusion. The use of SOD can enhance the survival of ischemic island skin flap.     

Protective effects of vitamins A and E pretreatment in venous ischemia/reperfusion injury

It has been suggested that venous ischemia is more injurious to tissue viability than is arterial ischemia of equivalent duration. The precise mechanism of tissue damage due to venous ischemia is still not well-determined. Current research has shown that it is multifactorial, and that lipid peroxides, prostanoid metabolism, and a free radical mechanism are the major contributors. Vitamins A and E are lipid-soluble vitamins that have been suggested to be successful in the treatment of arterial ischemia/reperfusion injury due to their antioxidant properties. In the present study, the authors examined the protective effects of vitamins A and E pretreatment on reperfusion injury induced by venous occlusion of rat epigastric island flaps based on an epigastric artery and vein pedicle. In the first part of the study, to determine critical ischemia time, epigastric island skin flaps (3 x 6 cm) were elevated on their vascular pedicles in 40 male Sprague Dawley rats. Total venous occlusion of the skin flap was produced by ligating all draining veins and clamping the epigastric vein. Arterial inflow was left intact. Rats were randomly assigned to five groups (n=8) for 2, 5, 7, 9, and 10 hr of venous ischemia. Despite the occurrence of widespread reperfusion injury, reflow was established (p<0.005) at 9 hr. In the second part of the study, 20 rats were randomly divided into four groups (n=5). The effects of vitamins A and E following 9 hr ischemia/reperfusion injury were examined. Rats were pretreated with vitamin E, vitamin A and vitamins A+E for 5 days. At the end of the fifth day, each rat had undergone an epigastric island skin flap and venous occlusion, as described above. Mean surviving flap area (percent) and plasma lipid peroxides (TBARs), total thiol content (t-SH), glutathione peroxidase (Gpx), and superoxide dismutase (SOD) were determined for each rat. Results suggest that, in the prevention of venous ischemia/reperfusion injury, vitamin A and vitamin E are not effective when used as single agents; however, when used in combination, they significantly increase surviving flap area by a synergistic effect.     

Protective effects of monoclonal antibody to intercellular adhesion molecule-1 in venous ischemia-reperfusion injury: experimental study in rats

Flaps with venous occlusion have a decreased survival rate compared with arterial occlusion. It seems that several factors are involved in the etiology of total venous occlusion, including free radicals, edema, thrombosis, and reperfusion injury. In the present study, the authors evaluated the blockage of polymorphonuclear leukocyte endothelial adhesion by using a monoclonal antibody to the intercellular adhesion molecule 1 (ICAM-1) ligand to prevent venous ischemia-reperfusion injury in rat epigastric island flaps. A skin flap (3 x 4 cm) supplied by the superficial epigastric artery and vein was harvested unilaterally in 40 male Wistar rats. Total venous occlusion of the skin flap was achieved. Arterial inflow was left intact. Rats were randomly divided into four groups (n = 10). In Group 1; rats were intravenously pretreated with 0.5 ml of 0.9 percent normal saline 15 min before applying a venous clamp, and the flaps were subjected to 6 hr of venous ischemia. In Group 2; rats were intravenously pretreated with 0.05 mg of monoclonal antibody to the intercellular adhesion molecule 1 (0.20 mg/kg) in 0.5 ml of 0.9 percent normal saline 15 min before applying the venous clamp, and the flaps were subjected to venous ischemia as in Group 1. In Group 3; rats were pretreated as in Group 1, and the flaps were subjected to 8 hr of venous ischemia. In Group 4; rats were pretreated as in Group 2, and the flaps were subjected to 8 hr of venous ischemia. The flaps were assessed histologically and by measuring viable and non-viable areas on postoperative day 7. Flap measurements revealed that blocking the action of ICAM-1 IN VIVO by administering monoclonal antibody significantly attenuated ischemic injury after 6 or 8 hr of venous occlusion.     

Effect of scavengers of superoxide radicals in reducing ischemic injury of skin flaps

In recent years, an extensive attention has been paid to the effects of oxygen free radicals on tissue injury. Although some studies have been demonstrated that the oxygen free radicals are implicated in the pathogenesis of ischemic injury in island skin flaps, none of the experimental study has been domestically reported. The authors used the island flap of the rats as an animal model to investigate the effectiveness of free radical scavengers in preventing reperfusion injury after ischemia in island skin flap. The results suggest that the scavenger of superoxide radical--superoxide dismutase (SOD) which was directly injected into the flap before and after reperfusion increases the survival rate of the flap obviously (93.6% v.s. control 63.7%). This findings are consistent with the theory that the ischemic tissue injury is caused by the oxygen free radicals produced at the time of tissue reperfusion.     

Detection of free radicals in reperfused dog skin flaps using electron paramagnetic resonance spectroscopy: a pilot study.

This is a preliminary investigation of the usefulness of spin-trapping electron paramagnetic resonance (EPR) in detecting oxygen-derived free radicals after reperfusion of ischemic skin flaps. Fourteen island skin flaps approximately 12 cm long by 6 cm wide based on the caudal superficial epigastric vessels were isolated in seven dogs. Seven of the paired skin flaps were subjected to 4 hours of arterial and venous occlusion. The opposite skin flap on each dog served as a perfused control. Spin trapping-EPR was able to detect radical adducts in five of the seven reperfused skin flaps. Hyperfine splitting constants of the radical adducts from the blood of three of the flaps confirmed biologically derived free radical production. Contaminating EPR signals from free radicals created in the manufacture of plastics probably masked any biologically derived radical adducts in the remaining flaps. The authors conclude that EPR-spin trapping may be a valuable tool in the study of the importance of oxygen-derived free radicals in the failure of skin flaps subjected to transient ischemia.     

The effect of a free-radical scavenger, N-2-mercaptopropionylglycine, on the survival of skin flaps

Oxygen free radicals may have an important role in tissue injury, which occurs on reperfusion of previously ischemic skin flaps. Therefore, therapy directed against the toxic effects of reactive oxygen species may protect skin flaps from ischemia/reperfusion injury. Various scavengers of oxygen free radicals have previously been reported to be effective in ameliorating ischemia/reperfusion injury. In the present study, N-2-mercaptopropionylglycine (MPG), a free-radical scavenger, was evaluated for its effectiveness in limiting the extent of necrosis resulting from ischemia/reperfusion injury in rat skin. Island skin flaps were elevated in the abdomen and groin based on an inferior epigastric neurovascular pedicle. The venous drainage from the flap was occluded for 7 hours, and reperfusion was established. The majority of flaps in the control group exhibited complete necrosis on Day 7 postoperatively. Treatment with systemic MPG (20 mg/kg of body weight) significantly improved flap survival from 22 to 71% (p less than 0.01) when administered at the time of reperfusion. However, MPG administered 1 hour after reperfusion did not influence the survival of the flaps. The results suggest that MPG may exert its beneficial effects on flap survival by scavenging oxygen free radicals formed at the time of reperfusion following prolonged ischemia.     

A biochemical study of acute ischemia in rodent skin free flaps with and without prior elevation

Elevation of a vascular island flap 24 hours before an ischemic insult (prior elevation) has been shown to significantly increase flap survival, and to decrease blood thromboxane levels, compared with acutely ischemic flaps. The current study considered whether prior elevation causes other biochemical alterations that could be beneficial for flap survival. Tissue levels of adenosine triphosphate (a major tissue energy store), superoxide dismutase (a major defense against free radicals), xanthine oxidase (an enzymatic source of free radicals), and edema were measured. Rat epigastric flaps, with or without prior elevation, had 10 or 12 hours of acute ischemia. Biopsies were taken at 0, 12, or 24 hours after reperfusion. Skin from flaps with no ischemia (control flaps) or control skin was harvested at the same times. Acutely ischemic flaps had significantly lower levels of adenosine triphosphate and less edema than those in prior elevated ischemic flaps after 12 hours of ischemia (both, p less than 0.05). Superoxide dismutase and xanthine oxidase did not vary significantly. It is not clear whether the increased adenosine triphosphate level in prior elevated flaps is the cause or the result of increased tissue viability. Prior elevation did not alter free radical mechanisms. Furthermore, prior elevation was beneficial for flap survival despite increased edema.     

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. © 1997 Wiley-Liss, Inc. MICROSURGERY 17:191–197 1996     

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.     

Prevention of injurious effect of free radicals to island skin flap with mannitol and anisodamine--an experimental study]

Rat abdominal skin flaps were subjected to total venous occlusion for 8 hours. Prior to and immediately following release of the vascular occlusion, mannitol, anisodamine or placebos were administered. Mannitol and/or anisodamine significantly enhanced island flap survival. LPO or water content was significantly lower in drug groups than in placebo groups. However, XOD was not affected by the drugs. These findings indicated that oxygen free-radical played an important role in post-ischemic flap injury.     

Effect of clamp versus anastomotic-induced ischemia on critical ischemic time and survival of rat epigastric fasciocutaneous flap

BACKGROUND: There are many models used to explore ischemic-related phenomena. The rat epigastric fasciocutaneous flap model is the one most commonly used. Critical ischemic time is the maximum ischemic insult that tissue can undergo and still remain viable. Experimentally, ischemia is induced either by clamping the vascular pedicle or by dividing the pedicle then performing microvascular arterial and venous anastomosis. We sought to determine what effect the different methods of inducing ischemia have on the critical primary ischemic time and, thus, flap survival. METHODS: A right 3 cm x 6 cm groin flap based on the inferior epigastric vessels was raised in each rat. Ischemic times of 4, 6, 8, or 10 hours were induced either by placing temporary occlusion clamps on each vessel of the vascular pedicle (island pedicle group) or by ligation and division of the pedicle with subsequent microvascular anastomosis (free flap group). Survival was assessed at 7 days. RESULTS: The primary ischemic time at which one half of free flaps are predicted to die was calculated to be 7.60 hours, compared with 6.09 hours for the island pedicle flaps (p<.05). CONCLUSIONS: Fasciocutaneous flaps undergoing ligation and anastomosis are more resistant to ischemia than are those undergoing clamping of the pedicle. Possible etiologic factors responsible for this experimental finding are discussed.     

Increased survival of island skin flaps by systemic heparin in rats

The effects of systemic heparin on the survival of island skin flaps have been evaluated. Island flaps measuring 3 x 6 cm were created in the abdomen/groin of rats based on an inferior epigastric neurovascular pedicle. The venous drainage from the flap was occluded for various lengths of time ranging from 5 to 10 hours in the control rats and from 8 to 15 hours in the heparin-treated group of rats. The rats in the treated group received 1 ml of heparin solution (300-400 U/kg) through the contralateral saphenous vein immediately prior to the onset venous occlusion and an equivalent dose every 4 hours during venous occlusion. The group of control rats received 1 ml of saline solution. Flap survival was evaluated daily for 7 days. The flaps of control rats demonstrated a gradual decline of the survival rate after 6 hours of venous occlusion and no survival after 10 hours of venous occlusion. The flaps of heparin-treated rats exhibited complete survival until 12 hours of venous occlusion and no survival after 13 hours of venous occlusion. Thus, systemic heparin prolonged tissue tolerance to venous occlusion from 10 to 13 hours. The flaps of control rats demonstrated a significant accumulation of inosine plus hypoxanthine to 260% of normal after 8 hours of venous occlusion. The flaps of heparin-treated rats exhibited little alteration of inosine plus hypoxanthine, and contained a greater concentration of glucose and lactate than the flaps of control rats after 8 hours of venous occlusion. The results demonstrate that systemic heparin can increase tissue tolerance to venous occlusion and improve significantly the flap survival following subsequent reperfusion.     

Hemoglobin-based oxygen carrier does not improve survival of ischemic rat island groin flaps.

Reducing reperfusion injury to skin flaps is an effective means to improve the survival of the flap. By enhancing oxygen delivery to the microcirculation within the flap, ischemia-reperfusion injury should be decreased, improving the flap's survival. This study evaluated the effects of a hemoglobin-based oxygen carrier (Oxyglobin) on the development of necrosis and survival of ischemic rat island groin flaps. Sprague-Dawley rats were randomly assigned to one of three treatment groups. A groin flap was elevated on each rat and subjected to 9 h of ischemia. Rats in group I were given an intravenous infusion of 0.9% saline prior to elevation of the skin flap. Rats in group II were given an intravenous infusion of Oxyglobin prior to elevation of the skin flap. Rats in group III were given a low-dose intravenous infusion of Oxyglobin following the 9 h of ischemia, just prior to reperfusion. The flaps were monitored for 7 days postoperatively for necrosis. The percentage of flap necrosis was recorded at the end of 7 days. All rats were euthanized at the completion of the study and the flaps were harvested for histopathological analysis. No significant difference was noted in the survival of the flaps or the degree of necrosis in the rats treated with Oxyglobin compared to the control group. Thus, pre-reperfusion treatment with Oxyglobin did not improve the percentage of flap survival, or the degree of severity of necrosis in rat groin flaps subjected to 9 h of ischemia.     

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.     

Effects of superoxide dismutase and allopurinol on the survival of acute island skin flaps.

We have demonstrated previously that oxygen-derived free radicals are important mediators of tissue injury following ischemia (total venous occlusion) and reperfusion in small (3 cm X 6 cm) island skin flaps in rats. In this study, we evaluated extension of this concept to regional ischemia in large (8 cm X 8 cm) acute island skin flaps which were constructed to exceed their sole blood supply via unilateral inferior epigastric vessels. Under normal (control) circumstances, a significant portion of the flap would undergo necrosis at the periphery, mimicking the corresponding clinical situation. Blocking the generation of superoxide radicals from xanthine oxidase with a single dose of allopurinol prior to flap elevation significantly improved the area of flap viability from 34 +/- 12% to 57 +/- 6% (p less than 0.01) in the random portion of the flap, contralateral to the source of blood supply. Similarly, the detoxification of superoxide radicals with a single dose of superoxide dismutase improved viability from 41 +/- 6% to 58 +/- 7% (p less than 0.01). Similar results were obtained when either of these agents were administered 60 minutes after flap elevation. These findings suggest that oxygen-derived free radicals play an important role in the development of tissue necrosis in the critical transition zone between well-vascularized and devascularized skin.     

Comparison of blood flow after warm and cool ischemia in island flaps: latissimus dorsi myocutaneous and epigastric flaps in the dog

Cooling has been reported to improve the survival of tissues used in free transfers. Very little has been written about blood flow in flaps following ischemia and about the effect of temperature on flow. In each of 4 dogs, 2 island latissimus dorsi myocutaneous flaps and 2 island epigastric axial flaps were made ischemic for one hour. Flaps were subjected to three temperature conditions during ischemia: warm ischemia, cool ischemia, and cool ischemia followed by rewarming prior to reestablishment of blood flow. Following ischemia, blood flow in the artery of the pedicle was measured using an electromagnetic flowmeter. Both latissimus dorsi and epigastric flaps subjected to warm ischemia experienced a reactive hyperemia, a period lasting several minutes during which blood flow increased severalfold over baseline flow. Cooling the latissimus dorsi flap had no major effect on this hyperemia. However, cooling the epigastric flap attenuated the hyperemic response observed in a warm flap. Both the peak flow attained and the rate of rise to peak flow were decreased. Rewarming the epigastric flap prior to clamp removal gave a hyperemia similar to that observed in the warm flap.     

Postconditioning attenuates ischemia-reperfusion injury in rat skin flap

Reperfusion injury by the abrupt restoration of circulation after the prolonged ischemia has been remained unsolved problem in the reconstructive microsurgery. We tested the hypothesis that a procedure of intermittent interruption of reperfusion, i.e., postconditioning (post-con) attenuates ischemia/reperfusion (I/R) injury of rat epigastric skin flap. A complete 4 hours of ischemia was generated by occlusion of the pedicle of dissected flap. The post-con procedure was started at the end of ischemia. A cycle of 15 seconds of full reperfusion, followed by 15 seconds of complete reocclusion was repeated six times (3 min of total intervention) prior to the unlimited reperfusion. Flap necrosis area of post-con group was compared with sham (no ischemic exposure) and control (4 hours of ischemia followed by full reperfusion without intervention) groups at postreperfusion day 5. Histology and MPO activities of flaps were evaluated. The post-con group showed significantly reduced flap necrosis at the end of 5 days of reperfusion compared with the control. Decreased inflammatory cell infiltration and MPO activity indicated post-con attenuated acute inflammatory reaction caused by I/R. This study reports for the first time that ischemic post-con effectively attenuates skin flap I/R injury. With further study, post-con may eventually be clinically applicable for the I/R injury as an "after-injury strategy."     

Superoxide dismutase and catalase in skin flaps during venous occlusion and reperfusion

We studied the enzymatic concentrations of superoxide dismutase (SOD) and catalase (CAT) in rabbit abdominal island skin flaps during 8 and 10 hours of venous occlusion followed by 4 hours of reperfusion. A correlation between such enzyme activity at the end of ischemia and flap viability was tested. The results show that the accumulation of blood and blood-derived substances cause enzyme activity to increase considerably during venous occlusion. The amount of increase was similar for both 8- and 10-hour venous occlusion. "True tissue enzyme activity" (corrected for the contribution of blood and blood-derived substances) remained constant during venous occlusion, suggesting that the availability of scavenging enzyme does not correlate with ultimate flap viability. During early reperfusion, however, both SOD and CAT activity decreased, suggesting that membrane damage leads to an increase in membrane permeability and to the loss of intracellular enzymes.