Evaluation of thrombolysis in a porcine model of chronic deep venous thrombosis: an endovascular model.

PURPOSE: The advancement of catheter-based interventions for vascular recanalization has underscored the need for an experimental animal model of vascular thrombosis that can be used for the evaluation of interventional therapies. In this model, a porcine model of deep venous thrombosis with a novel endovascular technique was described, and the efficacy of thrombolytic therapy with urokinase was evaluated. METHODS: An endovascular device that consisted of a tapered polytetrafluoroethylene graft attached within a self-expanding nitinol stent was delivered to bilateral common iliac veins in 20 pigs. Venous thrombosis occurred as a result of flow stasis created by the intrastent stenosis. Catheter-directed pulse-spray thrombolysis with urokinase (250,000 units) and heparin (5000 IU) was performed on one limb while the contralateral limb received control saline solution. Thrombolysis was performed in 1 hour (n = 4), 8 hours (n = 4), 3 days (n = 4), 7 days (n = 4), and 14 days (n = 4) after the stent-graft deployment. Venography and intravascular ultrasound were used to evaluate the efficacy of thrombolysis. Light microscopy was used for histologic analysis of the thrombus. RESULTS: Complete thrombolysis was achieved in groups with deep vein thrombosis that were younger than 1 day. Angioplasty of the tapered stent-grafts in the completely thrombolysed iliac vein was successful in restoring venous flow. The efficacy of thrombolysis in 3-day, 7-day, and 14-day groups was 86% +/- 7%, 73% +/- 13%, and 42% +/- 23%, respectively. The thrombolytic efficacy was enhanced to 92% +/- 16% and 86% +/- 18% (P <.05) in 3-day and 7-day groups, respectively, when doses of the pulse-spray thrombolysis were doubled. Increased dosages of the thrombolytic agent, however, did not significantly enhance the thrombus dissolution in the 14-day group. CONCLUSION: The thrombolytic efficacy of urokinase correlated with the chronicity of deep venous thrombosis in our model. An increased dose of urokinase may be used to enhance the efficacy of thrombolysis in a 1-week-old thrombus.

Effects of nicotine and cotinine on porcine arterial endothelial cell function.

UNLABELLED: BACKGROUND. There has been a significant amount of research on the effects of nicotine on vascular biology; however, little is known about the effects of cotinine, the metabolic product of nicotine. This study used a novel vascular perfusion system to study the effects of nicotine and cotinine on the vascular endothelial cell function. METHODS: Porcine common carotid arteries were cultured in a novel vascular perfusion system with nicotine or cotinine or as controls. After 24 h, vessels were precontracted with norepinephrine and subsequently relaxed with acetylcholine. Vessel diameters were recorded and analyzed. After culture, samples were taken for en face, immunohistochemistry, and RT-PCR for eNOS. Porcine coronary arteries were incubated as controls or with nicotine or cotinine and tested on a myograph system to measure contraction and relaxation. RESULTS: Porcine carotid arteries treated with nicotine and cotinine showed a 27.2% and a 41.2% reduction in endothelial-dependent relaxation, respectively, as compared to control vessels (P<0.05). Rings of coronary arteries treated with nicotine relaxed similarly to control rings while cotinine-treated rings failed to relax to endothelial-dependent stimulation. RT-PCR for eNOS mRNA showed a 23. 2 and a 24.1% reduction in eNOS expression for nicotine- and cotinine-treated vessels, respectively (P<0.01). Additionally, immunohistochemical staining for eNOS showed less dense staining on nicotine- and cotinine-treated vessels as compared to controls. En face preparations showed normal endothelial cell morphology in all groups, but cell density decreased slightly in vessels treated with nicotine and cotinine. CONCLUSION: These results indicate that cotinine may have even more effect on the impairment of endothelial-dependent vasorelaxation than nicotine for the regulation of vessel tone in porcine carotid and coronary arteries.

A simple physiologic pulsatile perfusion system for the study of intact vascular tissue.

Perfusion vascular culture models may provide a useful link between cell culture models and animal culture models by allowing a high level of control over important parameters while maintaining physiologic structure. The purpose of this study was to develop and test a new vascular culture system for pulsatile perfusion culture of intact vascular tissue. The system generates a pulsatile component of flow by means of a cam-driven syringe and a peristaltic pump and compliance chamber. Cams were designed, constructed and tested to simulate canine femoral and common carotid artery flows. The mean pressure was adjusted between 60 and 200 mmHg without significantly affecting flow rate, flow waveform, or the pressure waveform. Porcine common carotid artery segments were cultured in this pulsatile perfusion system. The viability of vascular segments was tested after various culture times with a functional assay that demonstrated both smooth muscle cell and endothelial cell response to vasomotor challenge.

A porcine model of carotid artery thrombosis for thrombolytic therapy and angioplasty: application of PTFE graft-induced stenosis.

PURPOSE: To develop a porcine carotid artery thrombosis model for the evaluation of thrombolytic therapy and adjunctive angioplasty procedures. METHODS: Bilateral carotid thrombosis was induced in 16 pigs using endothelial crush injury followed by external polytetrafluoroethylene (PTFE, 5 x 2 cm2) wrap placement to create segmental carotid stenosis. Light microscopy was used to examine thrombus composition. Selective carotid catheterization was performed via a femoral approach. Two hours following carotid artery occlusion, a urokinase (250,000 IU) and heparin (1000 U) solution was pulse-sprayed in 1 carotid artery while the contralateral vessel received the control saline vehicle. The efficacy of thrombolytic therapy was assessed using carotid arteriography and intravascular ultrasound. The feasibility and technical efficacy of balloon angioplasty within the carotid stenosis model were also evaluated. RESULTS: Carotid artery occlusion occurred in 30 +/- 6 minutes following endothelial injury plus PTFE wrap placement. Histological examination of carotid arteries showed endothelial irregularity with fibrin-rich and platelet-rich thrombus. Urokinase was effective in recanalizing all occluded arteries (100%), while the control saline vehicle showed no effective thrombolysis (p < 0.001). Angioplasty was successful in restoring normal diameter in all arteries (100%). CONCLUSIONS: This carotid artery thrombosis model, which incorporates intimal injury with segmental stenosis, is simple to create and reproducible. It provides not only a model for the evaluation of thrombolytic therapy but also a practical training tool for adjunctive endovascular interventions.

Evaluation of thrombolysis and angioplasty in a porcine iliac artery thrombosis model: application of endovascular stent-graft-induced thrombosis.

PURPOSE: To develop a novel endovascular thrombosis model in the porcine iliac artery for the evaluation of thrombolysis and angioplasty. MATERIALS AND METHODS: A stent-inversion-graft (SIG) model combining either a 3-mm or 5-mm tapered expandable polytetrafluoroethylene (ePTFE) graft attached within a self-expandable, 10-mm nitinol stent was placed in the left common iliac artery via an ipsilateral common femoral artery approach in 24 pigs. When the iliac artery was thrombosed, urokinase (250,000 IU) plus heparin (1,000 units) were pulse sprayed via a contralateral femoral approach (n = 12). Saline pulse-spray was used as a control group (n = 12). Balloon angioplasty was performed to eliminate the stenotic tapered graft within the stent after successful thrombolysis. The efficacy of the thrombolysis was assessed with use of intravascular ultrasound (IVUS) and arteriogram. RESULTS: Both the 3-mm tapered and 5-mm tapered SIG models caused iliac artery occlusion in 22 +/- 5 and 41 +/- 9 minutes, respectively, after the deployment. Luminal patency was re-established successfully in all occluded arteries after urokinase infusion. Angioplasty was successful in eliminating the tapered stenosis and restoring the normal diameter in all iliac arteries treated with urokinase. Complete thrombolysis was achieved in both models treated with urokinase. CONCLUSION: This novel endovascular approach of inducing arterial thrombosis is simple to perform and reliably produces arterial thrombosis. The intraluminal stenosis is also amenable to angioplasty. This model is useful for the evaluation of antithrombotic treatment modality and adjunctive endovascular interventions.

Effects of homocysteine on smooth muscle cell proliferation in both cell culture and artery perfusion culture models.

BACKGROUND: Hyperhomocysteinemia is associated with increased risk for vascular disease. However, the pathogenic mechanisms of homocysteine are largely unknown. We evaluated the effects of homocysteine on smooth muscle cell (SMC) and endothelial cell proliferation in cell culture and on SMC proliferation of balloon angioplasty-injured arteries in a perfusion culture model. METHODS: Human and pig SMCs and endothelial cells were cultured with variable amounts of homocysteine for 72 h and the total cells were counted using a hemocytometer. Fresh pig carotid arteries were harvested from a local slaughterhouse and cultured in a newly designed artery perfusion culture system. Five groups of arteries (six per group) were cultured for 48 h under different conditions: normal control, balloon angioplasty injury alone, and injury with three different doses of homocysteine. Vessel viability was evaluated. SMC proliferation was assayed by bromodeoxyuridine (BrdU) DNA labeling. RESULTS: At concentrations equivalent to those in human hyperhomocysteinemia, homocysteine significantly stimulated both cultured human and pig SMC proliferation with a dose-dependent effect, while it inhibited cultured endothelial cell growth. Perfusion-cultured pig carotid arteries remained contractile in response to norepinephrine and relaxant to nitroglycerine, and viable cells were also isolated from the cultured arteries. SMC proliferation (BrdU index) showed significant differences among the groups. SMC proliferation was stimulated by vascular injury and further enhanced by homocysteine in a dose-dependent manner. The proliferative response occurred strongly on the luminal side of the vessel wall, with the effects tapering toward the adventitia. CONCLUSIONS: Homocysteine had a mitogenic effect on vascular SMCs and a cytotoxic effect on endothelial cells. This differential effect of homocysteine on vascular cells may represent a pathogenic mechanism of vascular lesion formation in patients with hyperhomocysteinemia.

A new perfusion culture system used to study human vein.

BACKGROUND: Cell culture studies, ring studies, and indirect physiologic studies are the predominant models used to study human vascular tissue. Such studies are limited in their capacity to permit physiologic single-factor changes or to provide the proper mechanical stress or extracellular matrix present in normal tissues. We present a newly devised organ culture system that addresses these issues and permits survival of intact segments of human vascular tissue in a perfused environment. Our experience culturing human saphenous vein with this system is detailed. METHODS: Perfusion culture chambers were designed and constructed in our laboratory. Excess saphenous vein segments were collected from coronary artery bypass graft cases at our hospital and then mounted into our perfusion culture system for 0, 24, 48, 72, or 96 h. Vasomotor assays, hematoxylin and eosin staining, bromodeoxyuridine staining, and factor VIII staining were performed to assess tissue survival. RESULTS: A total of 24 veins were cultured. Average vessel length was 5 cm. The vessels contracted and relaxed the following amounts: time 0 (6.7% contraction, 5.0% relaxation), 24 h (5.7%, 5.3%), 48 h (5.2%, 2.8%), 72 h (4.8%, 5.3%), 96 h (4.8%, 3.8%). Hematoxylin and eosin staining, bromodeoxyuridine staining, and factor VIII staining support the viability of the tissue segments. CONCLUSION: A new perfusion organ culture system has been devised that permits survival of intact human venous tissue for periods up to 96 h. Studies that permit physiologic single-factor changes along with precise control of the hemodynamic environment are possible with this system.

An endovascular model of carotid stenosis for the evaluation of thrombolysis and angioplasty.

PURPOSE: To develop a porcine carotid artery thrombosis model using a novel stent-graft device to evaluate the efficacy of thrombolytic therapy and angioplasty procedures. METHODS: An endovascular device made from a tapered polytetrafluoroethylene graft inverted in a self-expanding nitinol stent was delivered to bilateral carotid arteries via a right femoral approach in 16 pigs. Carotid thrombotic occlusion ensued from flow stasis created by the intrastent stenosis. Via selective carotid catheterization from a femoral approach, urokinase (250,000 IU) was pulse-sprayed in one carotid artery while a control saline solution was infused in the contralateral vessel; delivery times were 1 hour, 8 hours, 3 days, or 6 days after carotid occlusion (4 animals per time period). After thrombolysis, balloon angioplasty was performed to maintain carotid patency. Arteriography and intravascular ultrasound were used to evaluate the efficacy of thrombolysis. Light microscopy was used for histological analysis of the thrombus. RESULTS: Carotid artery occlusion occurred in 15+/-8 minutes after stent-graft placement in all animals. Urokinase was effective in recanalizing all occluded arteries in the 1-hour, 4-hour, and 3-day groups (100%) but was effective in only 2 of 4 animals in the 6-day group (p < 0.05). Overall thrombolytic efficacy was 78%+/-7%. Control saline solution showed no thrombolytic effect (p < 0.001). Angioplasty successfully restored normal luminal diameter in all fully lysed arteries (100%). Histological analysis showed fibrin-predominant thrombus with a varying degree of platelet deposition. CONCLUSIONS: This endovascular approach, which creates a carotid stenosis using this novel stent-graft device, is reliable in producing carotid thrombosis. In our model, thrombolytic therapy was effective in restoring luminal patency, and the intraluminal stenosis is amenable to balloon angioplasty. The model is useful for the evaluation of antithrombotic therapy and adjunctive endovascular interventions.

Acute occlusion of the abdominal aorta.

BACKGROUND: Acute aortic occlusion most commonly results from aortic saddle embolus or thrombosis of an atherosclerotic abdominal aorta. The purpose of this study was to review the experience at a university hospital to better define the diagnosis and management of this uncommon process. METHODS: A retrospective chart review was performed from patients admitted to Emory University Hospital with acute occlusion of the abdominal aorta from 1985 through 1997. RESULTS: Thirty-three patients were identified. In group EMB (n = 16), occlusion was due to saddle embolus. In group IST (n = 17), occlusion was attributed to in situ thrombosis of a severely diseased aorta. Operative procedures performed included transfemoral embolectomy (15), aorto-bifemoral bypass (9), axillobifemoral bypass (5), fasciotomy (3), and thrombolysis (1). The in-hospital mortality rate was 21% (31% EMB, 12% IST), and morbidity was significant and included mesenteric ischemia (6%), bleeding complications (9%), subsequent amputation (12%), renal failure (15%), recurrent embolization or thrombosis (21%), and cardiac complications (42%). CONCLUSIONS: Acute aortic occlusion has tremendous morbidity and mortality even with optimal surgical care.

Local anesthesia for above knee femoropopliteal bypass: an alternative technique to endoluminal bypass grafting.

BACKGROUND: The majority of patients with peripheral vascular disease have associated medical illnesses that contribute to a substantial incidence of perioperative complications. Some of the these complications may be related to the choice of anesthetic used. An ideal anesthetic would provide good analgesia, have few complications associated with its use, including minimizing hemodynamic instability, and provide an adequate surgical environment. MATERIALS AND METHODS: Ten patients with an occluded superficial femoral artery and claudication or rest pain were selected. Eligibility criteria were a non-obese thigh and an above knee popliteal segment of at least 10 cm. Lidocaine local anesthesia with systemic sedation was employed. Above knee femoropopliteal bypasses were performed in all patients. Intraoperative fluid volume, anesthetic dose and perioperative morbidity were recorded. RESULTS: All patients tolerated the procedure well. The operating environment was excellent. Intraoperative fluid requirements were less for patients receiving local anesthesia as compared with a similar group of patients undergoing above knee femoropopliteal bypass receiving regional anesthesia (mean 1750 ml versus 3386 ml). The mean dosage of lidocaine (0.5%) was 367 mg over a mean of 116 min operating time. All patients ambulated within 8 hours. There was no perioperative morbidity, mortality or graft occlusion. CONCLUSION: This technique is easy to perform and further reduces the systemic magnitude of anesthesia, while providing excellent perioperative analgesia and a satisfactory surgical environment. It may be ideal for the high risk patient, as intraoperative fluid volume requirements are reduced. In an era where endoluminal bypass grafting is being increasingly advocated, this technique retains the benefits of bypass grafting while possibly reducing the physiological insult.

Synthesis of N-(4'-pyridoxyl)sphingosine and its uptake and metabolism by isolated cells.

N-(4'-pyridoxyl)sphingosine was synthesized and characterized as a stable compound for specialized delivery of a bioactive lipid. It was found to be facilely taken up by hepatocytes although by a mechanism more typical for lipids than the one used by natural vitamin B6. Some of the N-(4'-pyridoxyl)sphingosine was metabolically acted upon inside the cell to release pyridoxal 5'-phosphate and sphingosine, but formation of pyridoxal 5'-phosphate from the synthetic compound was poor compared with natural vitamin forms of B6, which may partly be due to entrapment within cell membranes and to constraints at the level of cytosolic pyridoxal kinase which is responsible for phosphorylation of the vitamin. Unlike the parent long-chain base, the B6 conjugate was not particularly cytotoxic. Furthermore, the compound was neither an activator nor inhibitor of the respiratory burst of human neutrophils. These findings identify N-(4'-pyridoxyl)sphingosine as an interesting tool for studies of the cellular transport, metabolism, and functions of both vitamin B6 and sphingosine.