Verapamil enhances the survival of primary ischemic venous obstructed rodent skin flaps.

The effect of verapamil, a calcium channel blocker, on the survival of skin flaps subjected to primary venous obstruction was studied. Skin flaps 9 x 4 cm, which are axial patterns with random extension, were elevated in Sprague-Dawley rats. A microvascular clamp was placed on the vein alone for 8 hours. Group 1 received verapamil (0.3 mg/kg) before flap elevation and before clamp release; group 2 received saline on the same schedule. Group 3 received verapamil (0.3 mg/kg) as above, plus every 8 hours for 5 days postoperatively. Group 4 received saline on the same schedule. There was no difference in survival between groups 1 and 2. Group 3 had 100% improvement in the flap survival compared with group 4 (78% vs 37%). Verapamil, if administered for the duration of the experiment, significantly increased flap survival.

The dorsal rat flap: a discussion of the model and the salutary effect of cimetidine on flap survival.

Failure of skin flaps remains a significant clinical problem. The dorsal rat flap, a reliable experimental model, was used to test the efficacy of cimetidine in treating a failing flap. Flaps were elevated in 45 rats divided into three equal groups. Group 1 was a saline control group, Group 2 received cimetidine 250 mg/kg three times a day for 7 days postoperatively, and Group 3 received cimetidine for 1 day before surgery, and then as in Group 2. Necrosis was assessed on the seventh postoperative day. Group 2 had 31.1 +/- 1.3 (mean % +/- SEM) necrosis, significantly better than saline control animals (p less than 0.01) and pretreated animals (p less than 0.05). These results suggest the usefulness of cimetidine in ischemic flap surgery.

Effects of external beam irradiation on the TRAM flap: an experimental model.

The rat model of the transverse rectus abdominis musculocutaneous (TRAM) flap was used in the present study to determine the effects of external beam radiation on myocutaneous flap histology and pathophysiology. A total of 57 adult Sprague-Dawley rats underwent a TRAM procedure. A pilot study with 17 animals was first performed to determine proper radiation dosages, and the remaining 40 rats were then used in the definitive study. In half of the definitive study group, the flaps were subjected to fractionated doses of external beam radiation, whereas the other half served as controls. Six weeks after the last radiation dose, all animals were killed and the flaps were harvested for mechanical assessment and histopathologic evaluation. All TRAM flaps survived in both groups. The irradiated and nonirradiated flaps were minimally distinguishable in viscoelastic properties, as well as by histopathologic examination. Growth of the flap in the irradiated animals was significantly diminished (48 percent average surface area increase in irradiated flaps, versus 92 percent increase in nonirradiated flaps, p < 0.05). These findings suggest that the myocutaneous flap is relatively resistant to some of the known adverse affects of radiation on living tissues.

Age and tolerance to secondary ischemia in rat epigastric flaps.

The use of microvascular procedures is increasing as the population continues to age. The purpose of this study was to observe the survival of skin flaps after ischemic injury. Skin flaps (n = 50) underwent either 3 hours of primary (1 degree) or secondary (2 degrees) venous occlusion in young (2-3 mo) and old (18-22 mo) rats. Skin flap survival was assessed on postoperative day 7. Survival rates for young and old after 3 hours of 1 degree ischemia was 100% and 90% (ns). Survival rats for young and old after 2 degrees ischemia were 67% and 47% (ns).

Amelioration of secondary ischaemic injury by perfusion with University of Wisconsin (UW) solution in rat skin flaps.

This study was designed to observe the effect of perfusion with University of Wisconsin (UW) preservation solution on skin flap survival following secondary ischaemia caused by venous obstruction in rats. An epigastric flap model was used. Saline-perfused flaps exhibited no significant improvement in survival compared to untreated animals (NS). Skin flaps perfused with UW solution, however, had a significant increase in survival to 40% (8/20) (p < 0.01) when perfused before the onset of primary ischaemia and 30% (p < 0.05) when given before the onset of secondary ischaemia. These results show that UW solution improves skin flap survival, presumably through preservation of the microvasculature.

Deleterious effect of urokinase used to treat experimental intra-arterial thiopental injection injuries.

UNLABELLED: Inadvertent arterial drug injections continue to be an important source of morbidity. Although the clinical picture of thiopental injection has been well defined over the past 50 years, there is still much controversy concerning pathophysiology and treatment regimen. Recently, a case report showed the efficacy of urokinase in treating this problem. The current study used the reliable ear model to study more closely this phenomenon. Rabbits were divided into four groups. Ears in Group 1 rabbits (n = 10) received an intra-arterial thiopental (15 mg/kg) injection. Group 2 rabbits (n = 10) received thiopental followed by a 1-ml saline injection 15 minutes later. Group 3 rabbits (n = 10) received thiopental followed by 50,000 U of urokinase. Finally, Group 4 rabbits (n = 4) received an intra-arterial injection of saline alone. Necrosis was evaluated 2 weeks later and expressed as a percentage. Student's t tests were used to evaluate data significance. RESULTS: Group 1 (thiopental alone) and Group 2 (thiopental and saline) rabbits had significantly more necrosis than Group 4 (saline alone) rabbits, 21.2% and 17.5% versus 0% (p less than 0.001 for both). Group 3 (thiopental and urokinase) rabbits had significantly more necrosis (46.5%) than Groups 1 and 2 rabbits (p less than 0.001 for both). CONCLUSION: From this study, we found that treatment of intra-arterial thiopental injection injuries with urokinase was of no benefit, but more importantly, it increased tissue necrosis by approximately 100%. Clinical use of this treatment is to be discouraged until underlying mechanisms are better defined.

Further investigation of secondary venous obstruction.

The first ischemic insult a tissue suffers is primary (1 degree). A second ischemic episode, such as thrombosis after free tissue transfer may be regarded as secondary (2 degrees) ischemia. The current study investigated 2 degrees ischemia in rodent epigastric flaps. Flaps were elevated in 50 Sprague-Dawley rats: group 1 had 5 hours 1 degree venous ischemia induced by placement of microvascular clamps; group 2 was like group 1, except venous continuity was re-established by venous anastomosis after resection of the venous segment previously microclamped; group 3 had 15 minutes of 1 degree ischemia, 24 hr later 5 hr of 2 degrees venous ischemia was induced by placement of microvascular clamps; group 4 was like group 3, except the venous segment was excised. Necrosis was evaluated on postoperative day 7. Both secondary ischemic groups had significantly less flap survival than the corresponding primary ischemic groups (P less than 0.001 for both). Resection of a portion of the vein and subsequent microanastomosis did not reduce flap survival (NS). Secondary venous ischemia of 5-hr duration is poorly tolerated by rodent skin flaps. There was no difference in flap survival in those flaps whose veins were clamped for 5 hr compared to those flaps whose clamped venous segments were resected and re-anastomosed.

Secondary ischemic tolerance improved by administration of L-NAME in rat flaps.

Nitric oxide (NO) under basal conditions is an important regulator of vascular tone. Under ischemic conditions, however, NO can combine with superoxide anion to produce the damaging hydroxyl free radical. The current project observes the effect of inhibiting NO production (L-Nitro-amino-methyl-arginine, L-NAME) on flaps rendered ischemic by secondary (2 degrees) venous obstruction. Eighty rats had 3 x 6 cm skin flaps based on the epigastric vessels. Primary (1 degree) ischemia was produced by arteriovenous occlusion for 2 hours; (2 degrees) venous ischemia was induced by clamping the vein, alone for either 3 or 5 hours. Thirty minutes prior to 2 degrees ischemia, rats received either L-NAME (30 mg/kg) or saline buffer. Flap survival was assessed 7 days later and Chi-square analysis was used. At 3 hours of ischemia, treatment improved survival from 55% to 85% (P < 0.05). Treatment also improved survival at 5 hours of ischemia from 5% to 35% (P < 0.04). Although under resting conditions, NO is a potent vasodilator, during 2 degrees venous obstruction it may contribute to flap necrosis.

Thrombogenic effects of a nonthrombin-based fibrin sealant compared with thrombin-based fibrin sealant on microvenous anastomoses in a rat model.

The efficacy and safety of tissue adhesives needs to be clearly defined. A thrombin-based preparation of fibrin sealant has recently been shown to have deleterious effects on microvascular anastomoses in an animal model. The authors found that fibrin sealant constructed with a high concentration of bovine thrombin (1,000 IU per milliliter) was detrimental to microvascular patency when applied to the anastomosis in a rat free flap model. The microvenous anastomosis had the highest rate of thrombosis and failure in this model. A nonthrombin-based fibrin sealant has recently become available for experimental investigation. This study examined the thrombogenic effect of this nonthrombin-based fibrin sealant on microvenous anastomoses in a rat free flap model compared with the effect of traditionally prepared fibrin sealant with varying concentrations of thrombin. The conclusions reveal that flap survival with application of the nonthrombin-based fibrin sealant to the anastomosis was comparable with flap survival of the control animals. Flap survival with application of the traditionally prepared thrombin-based fibrin sealant was also comparable with flap survival of the control animals when a concentration of 500 IU per milliliter of thrombin was used. However, flap survival decreased significantly (p <0.005) when a concentration of 1,000 IU per milliliter of thrombin was used in the construct of the fibrin adhesive. These results support the previous findings of the harmful effects of thrombin when used in high concentrations and applied to the microvenous anastomosis of this free flap model. Moreover, this initial investigation with a nonthrombin-based fibrin sealant did not show any deleterious effects on the microvenous anastomosis compared with control animals.

Timing relationships for secondary ischemia in rodents: the effect of venous obstruction.

In a previous study, timing relationships were studied for flaps subjected to secondary ischemia by total pedicle interruption. In the current paper, using a rodent epigastric flap, a similar study for flaps subjected to secondary ischemia by venous obstruction was performed. These conditions were designed to mimic a venous thrombosis following flap transfer, as would be performed clinically. In Experiment 1, the time interval between primary and secondary ischemia was varied. When the interval was 72 hr, flaps with secondary ischemia had similar survival to those with primary ischemia. However, when the time interval was 24 hr, flap survival after secondary ischemia was significantly worse than after primary ischemia (p less than 0.01). In Experiment 2, the duration of primary ischemia was varied (15 min, 30 min or 1.5 hr), prior to a fixed interval between primary and secondary ischemia and 5 hr of secondary ischemia. These conditions produced significantly more necrosis than 5 hr of primary ischemia. Thus, even short periods of primary ischemia may have detrimental effects on flap survival after a subsequent period of secondary ischemia. This may have important clinical ramifications.

The beneficial effect of dextran on anastomotic patency and flap survival in a strongly thrombogenic model.

Dextran lowers the probability of thrombus formation, by reducing platelet aggregation and adhesion and by increasing fibrinolysis. All studies to date using dextran for microvascular reconstruction have examined only short-term (1 to 2 hr) patency in isolated vessels. The current study used an established thrombotic model (inverted sleeve interposition graft), to investigate the effect of dextran on the long-term survival of pedicle flaps. A6- x 3-cm epigastric flap was elevated. A 2-mm inverted sleeve interposition graft was placed on the artery side of the pedicle by microvascular techniques. One group received dextran (17 cc/kg) 2 hr preoperatively and every 6 hr postoperatively for the next 72 hr. A control group received saline on the same schedule. Survival was assessed on postoperative day 7, at which time necrosis was obvious. Several parameters (clotting studies and electron microscopy) were studied to characterize the phenomenon more clearly. Only 25 percent of saline-treated flaps survived (2/8), while all dextran-treated flaps survived (7/7) (p less than 0.02, Fisher exact test). Thus, dextran allowed flaps to survive by preventing thrombus formation, despite a strong thrombogenic focus.

Acute thrombogenic effects of fibrin sealant on microvascular anastomoses in a rat model.

Topically applied bioadhesives and hemostatic agents have gained wide acceptance in various surgical endeavors. However, the effect of thrombin-based fibrin sealant (fibrin glue) when applied to microvascular anastomoses has not been evaluated thoroughly. Although fibrin sealant has been used directly on vascular anastomoses in macrovascular surgery, there has been little exploration into the utility and potential complications when used in the microsurgical setting. This study explored the influence of fibrin sealant containing increasing concentrations of bovine thrombin on microvascular anastomoses in a rat epigastric free flap model. The survival of the free flap in this model appeared to be inversely proportional to the concentration of thrombin in the fibrin sealant. When thrombin alone was applied to the anastomoses, the rate of thrombosis was the highest. Venous anastomosis was the most sensitive to the deleterious effects of topically applied thrombin.

Effect of fibrinogen and thrombin concentrations on mastectomy seroma prevention.

Seroma formation remains a significant clinical problem which increases morbidity and hospital costs in patients undergoing mastectomy operations. This study evaluated the effect of varying the concentrations of fibrinogen and thrombin in fibrin sealant on successfully preventing seroma formation in a rat model. After axillary dissection, control animals (Groups I and II) had 1 ml of either normal saline or thrombin (100 U/ml) applied to the axilla while treated animals (Groups III-VIII) had increasing concentrations of 0.5 ml of fibrinogen (25, 50, or 100 mg/ml) and 0.5 ml of thrombin (10 or 100 U/ml) applied. Seroma volumes (means +/- standard deviation) were measured on Postoperative Day 5. They were largest in Group I (3.1 +/- 1.9 ml, n = 13) and Group II (3.9 +/- 2.7 ml, n = 15) and then decreased from a high in Group III (2.5 +/- 2.4 ml, n = 15) using fibrinogen, 25 mg/ml, and thrombin, 10 mg/ml, to a low in Group VIII (0.8 +/- 1.0 ml, n = 15) using fibrinogen, 100 mg/ml, and thrombin, 100 U/ml. Analysis of variance revealed a statistically significant difference in the mean seroma fluid volumes between the groups (P = 0.0021), while Scheffe's comparison showed a specific significant difference (P = 0.028) between the thrombin control (Group II) and the highest concentration fibrin sealant (Group VIII). The difference in seroma volumes for all control animals (Groups I and II), 3.5 +/- 2.4 ml, and all treated animals (Groups III-VIII), 1.7 +/- 2.1 ml, was highly significant by unpaired t test. (P < 0.0001). Thus, fibrin sealant was useful in reducing seroma formation in this rat model with the highest concentration of fibrinogen and thrombin appearing most effective. These data may be useful in guiding future clinical trials in humans.

Timing relationships for secondary ischemia in rodents: the effect of arteriovenous obstruction.

The first ischemic insult a tissue suffers is primary (1 degree) ischemia. A second ischemic episode, such as thrombosis after free tissue transfer, may be regarded as secondary (2 degrees) ischemia. Timing relationships were studied in a rodent epigastric-flap model. In a first experiment, the interval between 1 degree ischemia and 2 degrees ischemia was varied. Flaps which had 2 degrees ischemia 12 and 36 hr after the 1 degree episode, had decreased ischemic tolerance, compared with 1 degree ischemic flaps; 2 degrees ischemic flaps, after an interval of 72 hr, had an ischemic tolerance similar to 1 degree ischemic flaps. In a second experiment, the length of the 1 degree ischemia was varied. It was found that as little as 5 min of 1 degree ischemia significantly decreased the subsequent tolerance of 2 degrees ischemia. The possible clinical significance of these results is discussed.

Seroma prevention in a rat mastectomy model: use of a light-activated fibrin sealant.

Seroma formation following mastectomy and axillary dissection remains a common and significant problem contributing to patient morbidity and health-care costs. Previous data have suggested that fibrin sealant (FS), a biological adhesive, is capable of controlling lymphatic leakage and assisting with skin graft adhesion. In this study, the use of an experimental, light-activated FS under development by CryoLife (CFS) was evaluated in a rat mastectomy model in order to reduce seroma formation. CFS is a premixed form of FS, containing an inactivator that is reversed in the presence of light, causing sealant to form. In this model, rats underwent mastectomy and extensive dissection of the axillary lymphovasculature. Next, 1 ml of saline or FS was applied to the operative site and the wound was closed. Three groups of animals were evaluated 5 days postoperatively by measuring the volume (in milliliters) of seroma able to be aspirated from the surgical site. The saline control group (N = 20) had a seroma volume (mean +/- standard deviation [SD]) of 4.2 +/- 2.9 ml, while a form of CFS containing human fibrinogen (80 to 100 mg per milliliter) and human thrombin (20 U per milliliter) (N = 20) had a significantly smaller seroma volume of 1.1 +/- 1.6 ml (p < 0.001 analysis of variance). University of Virginia (UVA) FS, containing human fibrinogen (20 mg per milliliter) and bovine thrombin (500 U per milliliter) (N = 20), had a seroma volume of 2.0 +/- 1.6 ml (p < 0.01, compared to control; p > 0.2, compared to CFS). Thus, this form of CFS significantly reduced seroma formation compared to saline control and also appeared to result in a smaller fluid accumulation than with UVA FS, although this trend was not statistically significant. These data suggest that the use of CFS may help to reduce seroma formation in humans.