Microvascular repair following crush-avulsion type injury with vein grafts: effect of direct inhibitors of thrombin on patency rate

The aim of this study was to develop a standardized effective thrombogenic arterial anastomosis model, as usually encountered in clinical practice, and to offer a detailed evaluation of the antithrombotic effect of thrombin's direct inhibitors, antithrombin III and hirudin, as locally applied. Wistar rats were divided into four groups of 12 animals each. The carotid artery sustained a standardized crush-avulsion-type injury (groups B-D). A segment of the afflicted area was removed and replaced by a microvenous graft. Group A had no crush-avulsion injury inflicted; a microvenous graft replaced a simple resection from the center of the carotid artery. During microvascular anastomoses, normal saline (groups A and B), recombinant hirudin (group C), or antithrombin III (group D) were locally applied. Bleeding times were recorded, and patency tests were performed 20 min, 48 h, and 1 week after blood flow reestablishment. All grafts were harvested and examined histologically. Patency tests, 1 week postrevascularization, demonstrated that this experimental crush-avulsion injury model ensured low patency in group B (25%), whereas group A, which had no injury inflicted, achieved a 100% patency rate. The local application of hirudin and antithrombin III significantly increased bleeding times as well as the patency rate (92% and 75%, respectively) compared to group B. These findings indicate the efficiency of the experimental model and the potential use of thrombin's direct inhibitors in microvascular surgery.     

Microvascular repair with 1-mm polytetrafluoroethylene (PTFE) grafts: effect of recombinant tissue-type plasminogen activator (rt-PA) on the patency rate and healing process

The present study assesses the effect of recombinant tissue-type plasminogen activator (rt-PA) on the patency rate and healing process of microvascular polytetrafluoroethylene (PTFE) grafts. Wistar rats were used, divided into four groups of 25 animals each. After dissection of the carotid artery a segment of the vessel, 1 cm long, was resected and replaced by equal length graft. Two different type fibril length (30- or 60-microm) grafts of the same wall thickness (0.18 mm) were used. Normal saline or 3 mg/kg of body weight of rt-PA was applied locally in each group of different fibril length grafts. Patency tests were performed at 15 min and 4 weeks after blood flow was reestablished. All grafts were harvested and examined histologically. The results showed that local application of rt-PA improves patency statistically significantly in both types of fibril length grafts. Patency in 60-microm fibril length grafts was statistically significantly higher than that of 30-microm fibril length grafts, whether rt-PA was used or not. The use of rt-PA had no influence on the healing process of either type of graft.     

Microsurgical repair after crush-avulsion injury of the femoral vein in rats: prevention of microvascular thrombosis with recombinant human tissue-type plasminogen activator (rt-PA).

Vein thrombosis is often encountered in microsurgery, especially in the case of crush-avulsion injuries. The aim of this study was to investigate the effect of systemic administration of recombinant tissue-type plasminogen activator (rt-PA) on the patency of the femoral vein of the rat, which had previously sustained a crush-avulsion injury. The study consisted of 3 groups of male Wistar rats, 20 animals each. A standardized crush-avulsion injury model was used. After microvascular repair of the femoral vein, the animals received either normal saline (group A), heparin 100 U/kg body weight (group B), or rt-PA 3.5 mg/kg body weight (group C) systemically. Patency tests were performed at 20 minutes, 48 hours, and 1 week after blood flow reestablishment. According to our results, the patency rate of the rt-PA group was significantly higher than in both the control and heparin groups.     

A comparison of human recombinant tissue-type plasminogen activator and urokinase in microsurgical thrombolysis in the rat

A comparison was made between several characteristics of tissue-type plasminogen activator (t-PA) and urokinase (UK), potentially useful in microsurgery to restore perfusion to ischemic free-tissue transfers. An intraarterial infusion of one of the drugs (or normal saline [NS] as a control) was performed in conjunction with a rat femoral vein clot model. Both t-PA and urokinase were effective in lysing 100 percent of the venous clots on the side of infusion. This occurred in only 25 percent of controls (p = .0035). Thrombolysis on the contralateral side, a measure of systemic effect, occurred in 38 percent, 50 percent, and 13 percent of veins using t-PA, UK, and NS, respectively (t-PA vs. UK p = 1.0, t-PA vs. NS p = .28, UK vs. NS p = .14). Rethrombosis occurred in 13 percent and 25 percent of ipsilateral veins treated with t-PA and urokinase, respectively, and in one of the two veins that had resumed flow during saline infusion (t-PA vs. UK p = .30) Scanning electron microscopy was performed 4 hr and 48 hr after thrombolysis. No differences between thrombolytic agents was noted in terms of residual thrombus and vessel characteristics. The data suggest that t-PA and urokinase are effective, with no clear advantage of one agent over the other.     

Neurotoxic effects of exogenous recombinant tissue-type plasminogen activator on the normal rat brain

Thrombolytic therapy with intravenous and intra-arterial recombinant tissue-type plasminogen activator (rtPA) has been established for the treatment of acute ischemic stroke. However, tPA has also been suggested to have neurotoxic effects. The purpose of this study was to examine direct neurotoxicity of rtPA in vivo. The animals (Wistar rats) were divided to the following three groups: low-dose (15 micromol/L) rtPA group (n = 6); high-dose (30 micromol/L) rtPA group (n = 6); and control (physiological saline) group (n = 6). The rtPA solution was perfused into the cortex via a microdialysis probe. The volume of the lesion was quantified histologically by image analysis of the lesions. Blood-brain barrier (BBB) disruption was evaluated by intravenous injection of Evans blue, and injury to the basal lamina was evaluated by immunohistochemistry using an anti-laminin antibody. In the rtPA-perfused animals, a pale lesion was produced around the probe, and microscopically, neurons showed necrotic changes. The volume of the lesions increased significantly as the concentration of perfused rtPA was increased. Marked extravasation of Evans blue was observed, and laminin immunoreactivity of blood vessels in the rtPA-induced lesions was lost. These results suggest that rtPA promotes acute direct neurotoxicity and participates in disruption of the microvascular basal lamina to cause BBB disruption, thereby increasing edema formation.     

Microvascular thrombolysis to salvage a free flap using human recombinant tissue plasminogen activator

Nonsurgical thrombolysis using tissue plasminogen activator (t-PA) in conjunction with intravenous heparin infusion was successfully used to salvage a free flap with acutely decreased arterial inflow thought to be due to anastomotic thrombosis. Intravenous infusion (12 mg/hr) of t-PA was stopped after 15 min of therapy when spontaneous bleeding was noted at the groin flap donor site. Concomitant treatment with intravenous heparin resulted in rapid improvement of flap perfusion and ultimately complete survival without surgical intervention. This report suggests that in selected cases, target-specific anticoagulant therapy may be beneficial in salvaging microvascular reconstructions complicated by thrombosis. Further experience is warranted to define more clearly the role, the risks, and the extended benefits of this new mode of nonsurgical therapy.     

Studies on the prevention of postoperative thrombosis with a small dose of recombinant human tissue-type plasminogen activator

In order to prevent the thrombosis on foreign materials after the cardiovascular operation, the effect of a small dose of recombinant human tissue-type plasminogen activator (t-PA) on the prosthetic material was examined. A polyvinyl-chloride tube was inserted into the left atrium of 12 rabbits. In six animals (group I), t-PA of 2.6 mg was injected into the right atrium, and no drug was used in other six animals (group II) as the control. Polyvinyl-chloride tube was removed from each animal 24 hours after implantation. In the medicated group, no thrombus was found in the tube, however, heavy thrombus formation was recognized around the tube in the control group. No clotting abnormality was shown in the medicated group. The results of this study demonstrate that t-PA will have a remarkable benefit in the prevention of thrombosis on the foreign substance in the cardiovascular system.     

Refinement of the rat crush-avulsion femoral artery injury model

A model for studying arterial crush-avulsion injuries in the rat is described. The model uses a standardized crush of ～0.3 joules and a standardized avulsion. The crush is accomplished by gravity acceleration of a 400 g weight over a distance of 7.5 cm. The weight impacts an anvil over the artery distributing the force of the impact over a 10 mm length of the vessel. The avulsion is accomplished by a hemostat attached to a second, 120g weight. Vascular stasis for 90 sec after vessel repair in a 175–225 g rat will consistently result in an 80% anastomotic failure. The model allows evaluation of anticoagulant effects singly or in combination. Agents may be given either systemically by intravenous route or locally by intraarterial route. © 1993 Wiley-Liss Inc.     

Effects of recombinant tissue-type plasminogen activator on life-threatening acute pulmonary thromboembolism in a canine model: a comparative study of e6010 and alteplase.

We compared the effects a novel recombinant tissue-type plasminogen activator (rt-PA) E6010 with the more conventional therapeutic agent alteplase in a new canine model of life-threatening acute pulmonary thromboembolism (APTE). Fifty milliliters of autologous blood was obtained from anesthetized, adult mongrel dogs and mixed with 10,000 units of thrombin. The left pulmonary artery, pulmonary vein, and bronchus were ligated, and previously prepared blood clots were injected via the femoral vein until the mean pulmonary artery pressure (mPA) increased to 2.5-3.0 times over baseline mPA (control). E6010 (0.4 mg/kg) or alteplase (1.33 mg/kg) was administered to other animals following inducement of APTE. In control animals, 60 min after embolization, mPA increased from 13+/-3 mm Hg to 31+/-3 mm Hg (p < 0.0001), cardiac output (CO) decreased from 1.47+/-0.35 l/min to 1.15+/-0.39 l/min (p < 0.0001), and PaO2 decreased from 101+/-31 mm Hg to 65+/-20 mm Hg (p < 0.001). E6010 significantly reduced mPA from 31+/-3 mm Hg to 25+/-4 mm Hg (p < 0.0001) 30 min after administration. In the alteplase group, however, mPA did not significantly change. At 180 min following drug administration, further reduction of mPA was significantly observed in both treatments. CO and PaO2 did not improve after either treatment. The present study indicated that E6010 more rapidly reduced pulmonary hypertension in our APTE model. Because of its rapid action, E6010 might be a promising thrombolytic agent for treatment of APTE.     

The effects of recombinant tissue-type plasminogen activator (rt-PA) on canine cadaver lung transplantation

The intrapulmonary thrombi that form after the cessation of circulation are thought to be one of the major causes of graft function failure. We evaluated the effect of recombinant tissue-type plasminogen activator (rt-PA) in a canine cadaver lung transplant model. Donor dogs were killed by the intravenous administration of pancuronium bromide without heparinization, and left for 2h at room temperature. The donor lungs were then flushed with low potassium dextran glucose (LPDG) solution, being subjected to a total ischemic time of 3h. Following left lung transplantation, the contralateral pulmonary artery of the recipient dogs was ligated. In group 1 (n=6), chloride solution was administered from the main pulmonary artery for 90 min, commencing 15 min prior to reperfusion. In group 2 (n=6), 2.5 μg/kg per min of rt-PA, and in group 3 (n=6), 5.0 μg/kg per min of rt-PA, were continuously infused in the same manner as in group 1. Lung function, including arterial blood gases and pulmonary hemodynamics, was measured for 3h. The side effects of rt-PA were evaluated by measuring the prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen, alpha₂-plasmin inhibitor (α₂-PI), plasminogen, and fibrin/fibrinogen degradation product (FDP). All of the animals in the three groups survived throughout the observation period. The group 3 animals had significantly better gas exchange than the group 1 animals, and the pulmonary hemodynamics were significantly better in the group 2 and 3 animals than in the group 1 animals. The FDP levels in the group 2 and 3 animals were significantly higher than those in the group 1 animals, while the PT and APTT were significantly prolonged in the group 3 animals. These findings led us to conclude that rt-PA improves early lung function, particularly pulmonary hemodynamics.     

A successful lytic treatment of a perioperative pulmonary embolism necessitating resuscitation using recombinant tissue-type plasminogen activator

We report a patient with massive perioperative pulmonary embolism (stage IV) and cardiac arrest who was successfully treated with recombinant tissue plasminogen activator (rt-PA). Thrombolytic therapy was started with 25 mg rt-Pa over 15 min followed by another 25 mg over 45 min. During the next 5 h 50 mg were infused, and thereafter an additional 50 mg over 24 h. No major alterations in the plasmatic coagulation profile were noted. With the exception of moderate blood loss from one puncture site, no further bleeding complications occurred. The relevance of this new thrombolytic agent in the perioperative management of acute, life-threatening pulmonary embolism is discussed.     

Initiation of a fibrinolytic system in hepatic resection: The roles of tissue-type plasminogen activator and plasminogen activator inhibitor-1

The factors related to the initiation of fibrinolysis, especially with regard to the tissue-type plasminogen activator (tPA) and the plasminogen activator inhibitor-1 (PAI-1), were investigated in 15 patients who underwent hepatic resection, and the findings were compared between those with normal livers and those with diseased livers. It was found that tPA increased before hepatic division, whereas PAI-1 increased after hepatic division and reached a peak immediately following the operation. Plasminogen decreased during hepatectomy, reaching its lowest point on postoperative day 1, and increasing later. Decreased levels of both plasminogen and the ₂-plasmin inhibitor were considered to be partly due to plasmin formation in the blood. Patients with a diseased liver tended to have higher intraoperative values of euglobulin lysis activity and higher postoperative values of plasminogen activator, but significantly lower postoperative values of ₂-plasmin inhibitor than those with a normal liver. The results of this study suggest that activation of the fibrinolytic system occurs both during hepatectomy and in the early postoperative period, and that patients with a diseased liver are prone to develop hyperfibrinolysis during hepatectomy. Moreover, the increased levels of both tPA and PAI-1 can serve as one of the most sensitive markers for the vital reaction against surgical stress.     

Differential effects of acute and chronic wound fluids on urokinase-type plasminogen activator, urokinase-type plasminogen activator receptor, and tissue-type plasminogen activator in cultured human keratinocytes and fibroblasts

The effect of wound fluids collected from acute well-healing wounds and chronic nonhealing venous leg ulcers on the plasminogen activation system of keratinocyte and fibroblast cell cultures was studied in a simplified wound-healing model. Acute wound fluid was collected from donor sites of split skin grafts at different time points representing the progressive healing of the wound. Urokinase-type plasminogen activator, tissue-type plasminogen activator, urokinase-type plasminogen activator receptor, and plasminogen activator inhibitor 1 expression were studied. The methods used were immunocapture assay and immunocytochemistry. The results indicated that the later the acute wound fluid was collected, the greater the urokinase-type plasminogen activator and the lower the plasminogen inhibitor-1 level in treated cells. In contrast, the level of urokinase-type plasminogen activator receptor remained stable irrespective of wound fluid treatment. Immunostaining for urokinase-type plasminogen activator of acute wound fluid-treated cells showed a disseminated punctate pattern over the cell surface, but with chronic wound fluid, urokinase-type plasminogen activator was localized to focal contacts. Our findings support the view that in the acute wound environment the plasminogen activator system is proteolytically active and that in chronic leg ulcers urokinase-type plasminogen activator and urokinase-type plasminogen activator receptor may also be organized for cell adhesion and migration.     

The use of recombinant tissue-type plasminogen activator in a newborn with an intracardiac thrombus developed during extracorporeal membrane oxygenation

Extracorporeal membrane oxygenation (ECMO) support is often used to support infants and children with hemodynamic or respiratory failure. One of the major obstacles of safely treating a child with ECMO is balancing the risk of hemorrhage with the potential for thrombus development. Managing thrombosis in the setting of ECMO is challenging and has no defined algorithm. The use of recombinant tissue-type plasminogen activator (tPA) for thrombolysis has been previously described in cases where thrombi have developed despite adequate anticoagulation. In such situations, the risk of hemorrhage must be carefully balanced with the benefit of dissolving the clot and reestablishing flow. We present a case of an infant who required ECMO because of severe primary pulmonary hypertension and subsequently developed a right atrial thrombus adjacent to the ECMO cannula. The patient was treated with tPA with immediate improvement but had fatal intracranial hemorrhage almost 3 days after the tPA was administered. In this report, we review the current literature on tPA use during ECMO support and suggest a rational approach.     

Microvascular grafts: effect of diameter discrepancy on patency rates

This study examined the effects of diameter discrepancy between interpositional vein grafts within a range of 0.25 to 2 mm. One hundred rats underwent isolation of a 10 mm segment of the femoral artery, from which a 4 mm segment was removed. Venous grafts measuring 8 +/- 2 mm were interposed. The rats were categorized by their graft to artery diameter ratios. Patency was assessed upon completion and 4-6 days postoperatively. All surgical specimens were submitted to the pathology department for microscopic evaluation. Results showed Group I: graft to artery ratio 1:1, patency rate 90% (18/20); Group II: ratio 0.75:1, patency rate 80% (16/20); Group III: ratio 0.5:1, patency rate 60% (12/20), Group IV: ratio 0.25:1, patency rate 20% (4/20); Group V: ratio 2:1, patency rate 60% (12/20). It is concluded that 1:1 venous graft to artery ratios are optimal. When this is impossible, diameter discrepancies should be at least 0.75:1 for acceptable patency rates to be attained in microvascular grafting.     

纤溶酶原激活剂(t-PA)促进挤压伤大鼠坐骨神经再生

目的 观察组织型溶酶原激活剂（ｔ－ＰＡ）对大嫌坐骨神经挤压伤修复的影响,并对其在周转神经再生微环境中的作用进行探讨。方法 取３６只Ｗｉｓｔａｒ大鼠,手术暴露两侧的坐骨神经,均造成坐骨神经挤压伤,左侧为实验侧;右侧为对照侧。实验侧损伤处的坐骨神经外膜下注入２μｌ（５μｇ）ｔ－ＰＡ;对照侧则注入２μｌ的生理盐水。术后１４、２８、５６ｄ分别进行大体、电生理、组织学观察和形态学分析。结果 术后１４ｄ时ｔ－     

Microvascular salvage procedures with adjuvant antithrombotic therapy for restitution of patency in a rat model.

Thrombosis following microvascular anastomosis requires further surgical intervention, involving anastomotic resection and reanastomosis or interpositional vein grafting. This study was undertaken to investigate different methods of salvaging thrombosed vessels, using a rat-vein model of error-induced thrombosis. Vessels were reconstructed 4 hr after the onset of thrombosis, using one of three methods: Group 1--removal of the erroneously placed stitch; Group 2--anastomotic resection and re-anastomosis; and Group 3--resection and replacement with a vein graft. Adjuvant antithrombotic therapy was simultaneously evaluated, assessing the influence of systemic Iloprost or heparin. Patency rates at one day postoperatively were 0 percent, 12.5 percent and 37.5 percent for Groups 1, 2, and 3, respectively. Following Iloprost infusion, these rates increased to 25 percent, 25 percent, and 56.3 percent, respectively and, following heparin administration, to 50 percent, 68.8 percent, and 81.8 percent, respectively. Significant increases were found for vein grafting (Group 3), and for the heparin-treated subgroups using all three methods. Effective levels of both Iloprost and heparin were confirmed by increases noted in rat-tail bleeding times. Significant rates of recanalization by three days following one-day occlusion were found in Groups 1 and 2. These results support the application of vein-graft replacement for thrombosed veins, concurrently with systemic heparinization. This study further confirms the high rate of recanalization seen in thrombosed rat femoral veins.     

Surgical stress induces acute coronary release of tissue-type plasminogen activator in the pig

BACKGROUND: Tissue-type plasminogen activator (t-PA) is an endothelium derived key enzyme in the initiation of endogenous fibrinolysis. Acute regulated release of active t-PA occurs within minutes in response to threatening thrombotic vessel occlusion. The aim of this study was to investigate the impact of surgical stimulation on the kinetics of t-PA release in the coronary vascular bed in the pig. METHODS: In anaesthetised pigs (n=16), arterio-venous concentration gradients of t-PA, and plasma flows (retrograde thermodilution) were obtained across the coronary vascular bed before (control) and at 1, 3, 5 and 10 min after sternotomy. RESULTS: At control, no significant coronary net flux (release or uptake) of t-PA was observed, while sternotomy induced a rapid net release of total t-PA (132.6 ng x min(-1)), with an associated increase in active t-PA (93.6 ng x min(-1)). This response, evident already after 1 min, showed a peak at 5 min and returned towards baseline levels within 10 min. No concurrent alterations in aortic levels of active t-PA were found and haemodynamic variables were unaltered. CONCLUSION: The rapidly increasing and transient net coronary release of t-PA after sternotomy suggests that the endothelium actively promotes local endogenous fibrinolysis during surgery. Such events could reflect a dynamic responsiveness to protect the coronary circulation during stress.