In vivo model of intracranial stent implantation: a pilot study to examine the histological response of cerebral vessels after randomized implantation of heparin-coated and uncoated endoluminal stents in a blinded fashion

OBJECT: No animal model currently exists for the examination of time-dependent histological changes occurring in intracranial vessels after endoluminal stent placement. The authors' goal was to develop a reproducible in vivo model of stent implantation in intracranial vessels in dogs that was capable of demonstrating stent-related vascular changes after the implantation of coated and uncoated devices. METHODS: The authors implanted heparin-coated or uncoated stents in the basilar arteries (BAs) of 11 mongrel dogs. In a 12th animal, one coated stent was implanted in the BA and a second uncoated one was implanted in the distalanterior spinal artery. All the devices were oversized to induce intimal injury. Surviving animals were observed for 12 weeks, after which they underwent repeated angiography before planned death and removal of the brain. Histological studies and computer-assisted morphometric analyses were conducted on stent-treated and untreated sections of the BAs to assess the percentage of stenosis, neointimal proliferation, vessel injury, and inflammation. Perforating vessels partially covered by stent struts ("jailing") were studied for evidence of stenosis or occlusion. The pathologist, interventionists, histopathologist, histopathology technicians, and radiologist were blinded to the stent type. Seven stents (three uncoated and four coated) were removed from the six animals that were observed during the follow-up period. The mean neointimal proliferation was 0.42 mm2 in the group treated with uncoated stents and 0.18 mm2 in the group treated with heparin-coated devices (p = 0.04). Neointimal thickness was significantly increased in the group with uncoated stents (p = 0.04). The mean percentage of occlusion was less (12%) in the group with heparin-coated stents, compared with 22% in the group with uncoated devices (p = 0.07). When comparing results between the heparin-coated and uncoated devices implanted in the five animals that received a single stent only, greater differences (indicating a benefit from heparin-coated stents) were observed in neointimal area (p = 0.009), neointima/media ratio (p = 0.001), neointimal thickness (p = 0.002), and percentage of occlusion (p = 0.009). All brainstem perforating vessels covered by stent struts remained patent. CONCLUSIONS: This in vivo intracranial stent model was developed to assess proliferative and inflammatory responses to endoluminal stent implantation in the cerebrovasculature. The results indicate that a lower percentage of occlusion occurs 12 weeks after implantation of heparin-coated compared with uncoated stents.     

Small-caliber heparin-coated ePTFE grafts reduce platelet deposition and neointimal hyperplasia in a baboon model

PURPOSE: Intimal hyperplasia and graft thrombosis are major causes of graft failure. Heparin prolongs graft patency and inhibits neointimal hyperplasia in animal models. The purpose of this study was to evaluate the effect of a heparin-coated expanded polytetrafluoroethylene (ePTFE) graft on platelet deposition and anastomotic neointimal hyperplasia after aortoiliac bypass grafting in a baboon model. METHODS: Heparin-coated ePTFE grafts (4-mm diameter) were incorporated into exteriorized femoral arteriovenous shunts placed in five baboons. Platelet deposition was analyzed by measuring the accumulation of indium 111-labeled platelets on the grafts, with dynamic scintillation camera imaging. Eight adult male baboons (mean weight, 9.3 kg) underwent bilateral aortoiliac bypass grafting with ePTFE grafts (4-mm internal diameter). In each animal a heparin-coated ePTFE graft was placed in one aortoiliac artery, and an uncoated graft, which served as the control, was placed in the contralateral aortoiliac artery. All grafts were harvested at 4 weeks, and were analyzed quantitatively for neointimal hyperplasia at graft-vessel anastomoses. RESULTS: Early platelet deposition on heparin-coated grafts after 1 to 4 hours of ex vivo circuitry was significantly reduced. All the harvested aortoiliac grafts were patent at 4 weeks. There was a significant reduction in neointimal area at both proximal (0.26 +/- 0.11 mm(2)) and distal (0.29 +/- 0.14 mm(2)) anastomoses in the heparin-coated grafts, compared with proximal (0.56 +/- 0.18 mm(2)) and distal (0.63 +/- 0.21 mm(2)) anastomoses in the untreated control grafts (P <.05). In addition, neointimal cell proliferation assayed with bromodeoxyuridine (BrdU) incorporation was reduced in the graft neointima (3.47% +/- 0.43%) in heparin-coated grafts compared with the graft neointima (6.21% +/- 0.59%) in untreated control grafts (P <.05). CONCLUSIONS: Small-caliber heparin-coated ePTFE grafts significantly reduce platelet deposition and anastomotic neointimal hyperplasia and cell proliferation, without measurable side effects, in baboons. Surface coating with heparin in small-caliber ePTFE grafts is useful for improving prosthetic bypass graft patency. Clinical relevance: An autologous vein graft is the ideal bypass conduit in peripheral arterial reconstruction; however, many patients who undergo bypass grafting do not have adequate or available autologous vein graft. As a result surgeons often must rely on prosthetic grafts as an alternative conduit in arterial bypass procedures. Clinical outcomes with prosthetic grafts in peripheral arterial reconstruction are generally inferior to those with autologous vein bypass grafts, in part because of anastomotic neointimal hyperplasia. This study evaluated the effect of small-caliber heparin-coated expandable polytetrafluoroethylene (ePTFE) grafts in aortoiliac reconstruction in a baboon model. The study found that heparin-coated ePTFE grafts resulted in less intimal hyperplasia and less platelet deposition after implantation, compared with noncoated control ePTFE grafts.     

Evaluation of platelet deposition and neointimal hyperplasia of heparin-coated small-caliber ePTFE grafts in a canine femoral artery bypass model

INTRODUCTION: Bypass graft failure due to acute thrombosis and intimal hyperplasia remains a major challenge in small-diameter vascular prosthetic graft reconstruction. Heparin has been shown to prevent thrombus formation and inhibit intimal antithrombotic in animal studies. In this study, we evaluated the effect of small-caliber heparin-coated expanded polytetrafluoroethylene (ePTFE) grafts on platelet deposition and intimal hyperplasia in a canine model of femoral artery bypass grafting. METHODS: Nine adult greyhound dogs underwent placement of bilateral femorofemoral artery bypass grafts with ePTFE grafts (4 mm diameter and 7 cm long). In each animal, a heparin-coated ePTFE graft was placed on one side while a noncoated graft was placed on the contralateral side which served as the control. Platelet deposition was measured by autologous (111)indium-labeling and scintillation camera imaging analysis in 24 h. The graft patency was assessed at 4 weeks following the bypass. The effect of intimal hyperplasia was assessed with histological and morphometric analysis. RESULTS: Platelet deposition on the heparin-coated grafts at 24 h was significantly reduced by 72% as compared to controls (P = 0.001). The patency rate was 44% in control grafts and 89% in heparin-coated grafts. There was a significant reduction of graft intimal hyperplasia at both proximal (0.38 +/- 0.21 mm(2)) and distal (0.19 +/- 0.06 mm(2)) anastomoses in the heparin-coated grafts as compared with proximal (1.01 +/- 0.28 mm(2)) and distal (0.42 +/- 0.01 mm(2)) anastomoses in the untreated control grafts, respectively (P < 0.05). Heparin coating significantly reduced graft neointimal hyperplasia at patent graft anastomoses by 55-72% as compared to controls. CONCLUSIONS: These data demonstrate that heparin coating of ePTFE significantly reduced early platelet deposition and inhibited anastomotic neointimal hyperplasia. Moreover, small-caliber heparin-coated ePTFE graft significantly increased graft patency in a canine femoral artery bypass model. This may represent a promising treatment strategy for improving the clinical performance of small-caliber prosthetic vascular grafts.     

Neointimal hyperplasia occurring after carotid endarterectomy in a canine model: effect of endothelial cell seeding vs. perioperative aspirin

Neointimal hyperplasia of the arterial wall may occur after carotid endarterectomy. This proliferative lesion is a pathologic response of the injured arterial wall and may lead to progressive stenosis. We investigated the effect of endothelial cell seeding (ECS) or antiplatelet therapy with aspirin (ASA) on inhibition of this lesion in a canine model. Endarterectomies were performed in 160 carotid arteries; 46 endarterectomies were treated perioperatively with aspirin (325 mg per day), 34 were seeded with a high density (3 X 10(6)) of autogenous endothelial cells, and 80 were untreated control arteries. At selected time intervals, the patent arteries were perfusion-fixed and the cross-sectional area (measured in square millimeters) of neointimal hyperplasia was measured by means of digital planimetry. At 6 weeks, patency of the endarterectomized carotid artery was 88% in the ASA and ECS groups, in contrast to 35% in the control group (p less than 0.01). The cross-sectional area of neointimal hyperplasia was not significantly different in the ASA and the control groups at 6 weeks. However, the ECS group showed a marked reduction in neointimal hyperplasia at 6 weeks (p less than 0.01). This inhibition of neointimal hyperplasia after carotid endarterectomy by ECS may reflect accelerated luminal healing or a direct inhibition of smooth muscle cell proliferation in the injured arterial wall.     

Inflammatory responses involving tumor necrosis factor receptor-associated factor 6 contribute to in-stent lesion formation in a stent implantation model of rabbit carotid artery

OBJECTIVE: Inflammatory responses are considered to represent a unique property after stent implantation, and we previously demonstrated that inflammatory signaling involving tumor necrosis factor receptor-associated factor 6 (TRAF6) contributes to neointimal formation in a balloon injury model of rabbit carotid artery. The purpose of this study was to examine the role of TRAF6 in in-stent lesion formation after stent implantation in the rabbit carotid artery. METHODS: Rabbit carotid arteries were injured with a 2F Fogarty catheter, and 28 days later, the same arteries were implanted with a 3-mm-diameter Palmaz-Schatz stent. A dominant negative (DN) form of TRAF6 (pME-FLAG-T6deltaRZ5) was then transferred using a plasmid-based electroporation method. Its effects were evaluated compared with the findings in arteries treated with control plasmid (pME-FLAG). RESULTS: Immunostaining with anti-FLAG tag antibody showed that an expression plasmid vector containing the DN-TRAF6 sequence was successfully transferred to the arterial intima and media. Morphometric analyses revealed that the increase of intimal area in in-stent lesions was significantly inhibited by DN-TRAF6 14 days after stent implantation (DN-TRAF6 group, 3.01 +/- 0.25 x 10(5) microm2 vs control group, 4.25 +/- 0.23 x 10(5) microm2, P < .01), and the cell density was increased compared with that in the control group. In the DN-TRAF6 plasmid-treated vessels, cell replication was prevented in both the intima and media, and fewer leukocytes adhered to the luminal surface. Moreover, DN-TRAF6 suppressed macrophage infiltration, activation of proteases, and proteoglycan accumulation in the in-stent intima. CONCLUSIONS: These findings suggest that TRAF6 plays an important role in cell replication, inflammatory cell infiltration, protease activity, and extracellular matrix accumulation that contributes to in-stent lesion development.     

Inhibition of experimental neointimal hyperplasia by recombinant human thrombomodulin coated ePTFE stent grafts

OBJECTIVES: The goal of this study was to evaluate the ability of recombinant human thrombomodulin (rTM) to inhibit neointimal hyperplasia when bound to expanded polytetrafluoroethylene (ePTFE) stent grafts placed in a porcine balloon injured carotid artery model. METHODS: The left carotid artery of male pigs, weighing 25 to 30 Kg, was injured with an angioplasty balloon. Two weeks later either a non-coated standard ePTFE stent graft (Viabahn, 6 x 25 mm, W. L. Gore & Associates) or a rTM coated stent graft was implanted into the balloon-injured segment using an endovascular technique. Carotid angiography was performed at the time of the balloon injury, two weeks later and then at 4 weeks to assess the degree of luminal stenosis. One month after stent graft deployment, the grafts were explanted following in situ perfusion fixation for histological analysis. The specimens were then cross-sectioned into proximal, middle and distal segments, and the residual arterial lumen and intimal to media (I/M) ratios were calculated with computerized planimetry. RESULTS: rTM binding onto ePTFE-grafts was confirmed by functional activation of protein C and histopathology with immuno-scanning electron microscopy, backscatter electron emission imaging and x-ray microanalysis. All seven of the rTM coated stent grafts and six of the seven uncoated stent grafts were patent at the time of explantation. The mean luminal diameter of the rTM coated stents was 93% +/- 2.0% of the original diameter, compared with 67% +/- 23% (P = .006) in the control group. Histological analysis demonstrated that the area obliterated by intimal hyperplasia at the proximal portion of the rTM stent was -27% compared with the control group: (2.73 +/- 0.69 mm(2), vs 3.47 +/- 0.67 mm(2), P <.05). CONCLUSIONS: Neointimal hyperplasia is significantly inhibited in ePTFE stent grafts coated with rTM compared with uncoated grafts, as documented by improved luminal diameter by angiography and by computerized planimetry measurements of residual lumen area. These findings suggest that binding of recombinant human thrombomodulin onto ePTFE grafts may improve the long-term patency of covered stents grafts. CLINICAL RELEVANCE: Decrease of neointimal hyperplasia of the magnitude observed in this study could significantly improve blood flow and patency of small caliber prosthetic grafts. If the durability of these results can be confirmed by long-term studies, this technique may prove useful in preventing graft stenosis and arterial thrombosis following angioplasty or vascular bypass procedures.     

Effects of thromboxane synthetase inhibition on patency and anastomotic hyperplasia of vascular grafts

The efficacy of a thromboxane synthetase inhibitor (U-63,557A, Upjohn) in promoting early patency and inhibiting anastomotic intimal hyperplasia in ePTFE grafts was compared to that of acetylsalicylic acid (ASA) in a canine model. Animals were started on ASA 5 gr po qd (Group I, n = 12) or U-63,557A 10 mg/kg po bid (Group II, n = 12) 1 day before placement of bilateral 5-mm-i.d., 13- to 16.5-cm-long ePTFE aortoiliac grafts and continued on the medication for the 16-week study. Six dogs in each group received autologous endothelial cell-seeded grafts, while the other six received unseeded grafts. Patency was determined weekly by assessment of femoral pulses. At the conclusion of the study anastomotic intimal hyperplasia was measured on serial sections through the distal anastomosis using a computer-linked digitizer. In Group I the patencies of seeded and unseeded grafts were not significantly different, being 100 and 83%, respectively. Furthermore, luminal narrowing due to intimal hyperplasia was not significantly different being 9.1 +/- 7.6% (chi +/- SD) in seeded grafts and 8.8 +/- 8.1% in unseeded grafts. On the other hand, in Group II the seeded grafts had significantly improved patency when compared to the unseeded grafts (83% vs 33%, P less than 0.05) and less luminal narrowing (11.4 +/- 11.1% vs 21.9 +/- 19.5%, P less than 0.01). Although U-63,557A administration promoted patency of unseeded grafts compared to no antiplatelet medication (0% patency), it was significantly less effective than ASA in improving patency (P less than 0.05) and inhibiting luminal narrowing (P less than 0.01).     

A prospective evaluation of arterial intimal injuries in an experimental model

The management of arterial intimal defects remains controversial because of uncertainty concerning their natural history. We developed an experimental canine model to prospectively evaluate posterior wall intimal flaps in the superficial femoral artery. Arterial intimal flaps were constructed in 20 anesthetized dogs (40 arteries) and evaluated by arteriography, and angioscopy, and intravascular ultrasound. Postoperative patency rates at 1 (n = 20) and 3 weeks (n = 20) were compared with a control group of ten animals (n = 20, arteriotomy without intimal flap). Acute thromboses occurred in five experimental arteries with thromboses of eight additional experimental arteries at followup. Control patency was 100%, while experimental group patencies were 75% (p less than 0.05) at 1 week and 60% (p less than 0.009) at 3 weeks. All thrombosed arteries had intimal flaps with greater than 75% luminal stenosis. We conclude that intimal injuries cause arterial thromboses acutely and during subsequent followup. Intimal flaps with stenosis greater than 75% as determined arteriographically are at greatest risk for thrombosis. Angioscopy and intravascular ultrasound characterize arterial intimal defects and may delineate injuries requiring surgical or endovascular repair.     

Preservation of endothelial integrity and function in experimental vascular anastomosis with non-penetrating clips

BACKGROUND: Vascular repair with sutures is associated with disruption of the endothelial lining and subsequent thrombus formation on the intraluminal lesions. This experimental study was designed to determine whether the use of non-penetrating clips improved endothelial preservation. METHODS: In ten female pigs, 25-mm arteriotomies were made in both carotid arteries. The arteriotomies were repaired with jugular vein patches. On the left side, the repair was done with 1.4-mm titanium clips, and on the right side with two running 6/0 polypropylene sutures. Next, the aorta was divided and subsequently repaired with 2-mm clips in five of these pigs, and with two running 5/0 polypropylene sutures in the remaining five pigs. Endothelial function was studied at the anastomotic site in the carotid arteries by determination of endothelium-dependent and -independent relaxatory responses. Morphometric examination of the carotid arteries and inspection of the aortic endothelium were performed by means of scanning electron microscopy. RESULTS: Maximal endothelium-dependent relaxation to adenosine 5'-diphosphate was less in sutured than in clipped carotid arteries (P < 0.05), while there was no difference in maximal endothelium-independent relaxation to sodium nitrite. This result in clipped carotid arteries was not accompanied by less intimal hyperplasia. Screening of the aortic anastomotic line showed better preservation of endothelial architecture after clip anastomosis. Mean cross-clamp time for carotid patch repair was significantly less when using clips than with sutures. CONCLUSION: The use of non-penetrating clips for vascular anastomoses preserved endothelial function and structural integrity better than running sutures, although the degree of intimal hyperplasia was similar.     

The Effects of PPARgamma Agonist Rosiglitazone on Neointimal Hyperplasia in Rabbit Carotid Anastomosis Model

ABSTRACT: OBJECTIVE: Neointimal hyperplasia involving smooth muscle cell (SMC) proliferation, migration and extracellular matrix (ECM) degradation is an important component of atherosclerosis. It develops as a response to vascular injury after balloon angioplasty and vascular graft placement. Matrix metalloproteinases (MMPs) induce SMC proliferation, migration and contribute to intimal hyperplasia by degrading ECM. PPARgamma agonists inhibit SMC proliferation, migration and lesion formation. In this study, we aimed to investigate the effects of PPARgamma agonist rosiglitazone on neointimal hyperplasia and gelatinase (MMP-2 and MMP-9) expressions in rabbit carotid anastomosis model. Method: New Zealand white rabbits (n=13, 2.7-3.2 kg) were divided into placebo and treatment groups. Right carotid artery (CA) was transected and both ends were anastomosed. Treatment group (n=6) received rosiglitazone (3mg/kg/day/p.o.) and placebo group (n=7) received PBS (phosphate buffered saline, 2.5ml/kg/day/p.o.) for 4 weeks postoperatively. After the sacrification, right and left CAs were isolated. Morphometric analyses and immunohistochemical examinations for gelatinases were performed. Results: Intimal area (0.055+/-0.005 control vs 0.291+/-0.020 um2 anastomosed, p<0,05) and index (0.117+/-0.002 control vs 0.574+/-0.013 anastomosed, p<0,01) significantly increased in anastomosed arteries compared to control arteries from placebo group. However, in rosiglitazone-treated group, intimal area (0.291+/-0.020 PBS vs 0.143+/-0.027 rosiglitazone, p<0,05) and index (0.574+/-0.013 PBS vs 0.263+/-0.0078 rosiglitazone, p<0,01) significantly decreased. Furthermore, gelatinase immunopositivity was found to have significantly increased in anastomosed arteries from placebo group and decreased with rosiglitazone treatment. Conclusion: These results suggest that rosiglitazone may prevent neointimal hyperplasia, which is the most important factor involved in late graft failure, by inhibiting gelatinase enzyme expression.     

Tenascin-C-coated platinum coils for acceleration of organization of cavities and reduction of lumen size in a rat aneurysm model

OBJECT: Detachable platinum coils are widely used in the endovascular treatment of intracranial aneurysms. The use of coil placement produces a higher incidence of aneurysm recurrence compared with surgical clipping. To reduce the incidence of recurrence by promoting clot organization, the authors designed a platinum coil coated with tenascin-C (TNC), an extracellular matrix glycoprotein, and then histologically examined tissue responses. METHODS: Platinum coils were prepared by successive coatings with cationic polyethyleneimine and anionic heparin and then TNC or basic fibroblast growth factor (bFGF) was immobilized by affinity binding to the heparin. Six unmodified, six heparin-coated, six bFGF-coated, or eight TNC-coated platinum coils were inserted into ligated common carotid arteries (CCAs) of adult male rats, and CCA segments were harvested after 14 or 28 days. The percentages of organized areas occupying the luminal cavity in unmodified, heparin-coated, bFGF-coated, and TNC-coated groups were 4.8 +/- 4.6, 1.6 +/- 1.1, 17.9 +/- 10.7, and 93.4 +/- 6.9%, respectively. In addition, the mean lumen size in the TNC-coated group (0.35 +/- 0.23 mm2) was reduced to less than half that of the unmodified group (0.72 +/- 0.21 mm2). Immunohistochemical analysis revealed that alpha-smooth muscle actin-positive cells were a major cellular component of the organized tissue within the TNC-coated coils but not in the bFGF group. Collagen fibrils in the organized areas were also much thicker and denser with TNC-coated coils than with bFGF-coated coils. CONCLUSIONS: Placement of TNC-coated coils can remarkably accelerate organization of luminal cavities and reduce their volume, providing improved efficacy of these coils for endovascular embolization.     

Induction of vascular atrophy as a novel approach to treating restenosis. A review

Regardless of the type of arterial reconstruction, luminal narrowing (stenosis or restenosis) develops in approximately one third of the vessels. In the past, the focus of research has been on the mechanisms of stenosis (intimal hyperplasia, pathologic remodeling) and pharmacologic approaches to prevention. An alternative approach is to induce intimal atrophy after luminal narrowing has developed, thus limiting treatment to only those patients that develop a problem. This approach to treat established disease by reducing wall mass through induction of cell death and extracellular matrix removal would be particularly useful for treating stenosis in synthetic bypass grafts or stented vessels, in which intimal hyperplasia is the primary mechanism of stenosis. This approach may be applicable as well to other vascular proliferative disorders, such as pulmonary hypertension and chronic transplant arteriopathy. Proof of principle has been shown in experiments with antibodies to platelet-derived growth factor (PDGF) receptors that cause neointimal regression in baboon polytetrafluoroethylene (PTFE) grafts and with angiotensin-converting enzyme inhibitors that induce medial atrophy in hypertensive arteries. Possible molecular targets could include PDGF receptors, A20, and BMP4. Further studies are needed to determine the utility of such a therapeutic approach to vascular disease.     

Aspirin and dipyridamole decrease intimal hyperplasia in experimental vein grafts

Release from platelets of a factor mitogenic for smooth muscle cells is a postulated mechanism for the pathogenesis of vascular intimal hyperplasia. In this study the effect of antiplatelet therapy was evaluated. Aspirin (165 mg twice daily) and dipyridamole (25 mg twice daily) were administered to six rhesus monkeys and six were given placebo only. Bilateral vein bypass grafts were placed in the iliac arteries. In addition, to evaluate the relative contribution of adventitial dissection and intimal injury, on one side the carotid artery and femoral vein were stripped of adventitia and on the other side the intima of these vessels were injured by the single passage of an inflated balloon tipped catheter. Animals were killed after 16 weeks. In grafts relative luminal area was determined by a photographic gravimetric method at three standard locations. Femoral veins and carotid arteries were classified as histologically normal or as exhibiting hyperplasia. All vessels with adventitial stripping were normal. All vessels with intimal injury in the placebo group except one exhibited intimal hyperplasia compared to the drug treated group in which over half were normal. Relative intimal area was significantly less in grafts from drug treated animals at all three locations and luminal area greater in two. These data suggest that vascular intimal hyperplasia can be reduced by treatment with antiplatelet agents.     

Saratin, an inhibitor of von Willebrand factor-dependent platelet adhesion, decreases platelet aggregation and intimal hyperplasia in a rat carotid endarterectomy model.

PURPOSE: Post-carotid endarterectomy, thrombosis, and intimal hyperplasia may be decreased by the inhibition of platelet adhesion and activation. In this study, a novel agent, saratin, was used to inhibit platelet-to-collagen adhesion in a rat carotid endarterectomy model. Saratin is a recombinant protein isolated from the saliva of the medicinal leech Hirudo medicinalis, which is thought to act by binding to collagen, and inhibits von Willebrand factor-collagen interaction under conditions of increased shear and therefore, the adherence and activation of platelets at the vessel wall. Saratin has the advantage of being a nonsystemic, site-specific topical application. METHODS: A rat carotid endarterectomy model was used in which an open technique with arteriotomy and intimectomy was used. Saratin was applied to the endarterectomized surface of the carotid artery before arterial closure. End point measurements included platelet adhesion, thrombosis rate, intimal hyperplasia development, bleeding times, and platelet counts. Electron micrographs of carotid arteries were used for quantitative analysis of platelet aggregation and platelet counts. Intimal hyperplasia and thrombosis were assessed with computer-assisted morphometric analysis of elastin-stained carotid artery sections with direct measurement of the intimal hyperplasia area. RESULTS: The topical application of saratin significantly decreased platelet adhesion compared with controls at 3 hours after carotid endarterectomy (64 +/- 17 vs 155 +/- 33 platelets per grid, P = .05), and 24 hours after carotid endarterectomy (35 +/- 11 vs 149 +/- 37 platelets per grid, P = .0110), respectively. A percent luminal stenosis, as a measure of intimal hyperplasia, was significantly decreased with saratin application compared with controls (10.9% +/- 1.8% vs 29.8% +/- 6.8%, P = .0042). This decrease in intimal hyperplasia formation correlated with the inhibition of platelet adhesion. Thirty-three percent of control arteries were found to be thrombosed 2 weeks after carotid endarterectomy compared with a 0% thrombosis rate in the saratin-treated group (P = .0156). No increased bleeding was encountered along the arterial suture line in the saratin group. Bleeding times and systemic platelet counts were not found to change significantly in the saratin-treated rats compared with control rats at 3 and 24 hours after endarterectomy. CONCLUSION: Saratin significantly decreased platelet adhesion, intimal hyperplasia, luminal stenosis, and thrombosis after carotid endarterectomy in rats. Saratin did not increase suture line bleeding or bleeding times, and did not decrease platelet counts. Saratin may serve as a topical agent to be used for the site-specific inhibition of thrombosis and intimal hyperplasia after vascular manipulation.     

Adenoviral mediated uteroglobin gene transfer to the adventitia reduces arterial intimal hyperplasia

PURPOSE: The aim of this study was to investigate the feasibility of gene transfer of uteroglobin, a potent anti-inflammatory and immunomodulatory agent, via adenoviral mediated gene transfer to the adventitia in the mouse carotid ligation injury model and also to investigate the efficacy of uteroglobin in reducing neointimal hyperplasia. METHODS: Forty-five C57bl/6NHSD mice were anesthetized and left common carotid artery ligation was performed. Adenoviral vector encoding the uteroglobin gene (Ad.UG; 15 microl of 1.35 x 10(11) pfu/mL) was applied to the adventitia of the injured artery in 16 mice. In our control groups, 16 mice received adenoviral vector encoding the beta-galactosidase reporter gene (Ad.lacZ; 15 microl of 1.0 x 10(11) pfu/mL) and 13 mice received PBS only. Six mice from each group were sacrificed at 4 days for carotid artery protein extraction and Western blot analysis. The remainder were harvested at 30 days for histologic and morphometric analysis. The intima/media area ratios were calculated for each artery. The results were analyzed and compared using ANOVA and Bonferroni/Dunn post hoc testing. RESULTS: Two mice from the LacZ group and one from the PBS group died before the 30-day endpoint. Uteroglobin expression was demonstrated in the Ad.UG treated arteries by Western blot analysis. Morphometric analysis demonstrated a statistically significant reduction in the intima/media area ratio of Ad.UG treated carotids compared to controls. There was a reduction of intima/media ratio with Ad. UG treatment of 68% compared to Ad.lacZ treatment (P < 0.0001) and 62% compared to PBS treatment (P = 0.0006). There was no statistical difference between the control groups. CONCLUSION: Adenoviral mediated gene transfer via the adventitia is an effective mode of gene delivery. Adventitial uteroglobin gene transfer using an adenoviral vector induces uteroglobin protein production and significantly reduces neointimal hyperplasia in the mouse carotid ligation injury model.     

The effects of Toradol on postoperative intimal hyperplasia in a rat carotid endarterectomy model: laboratory research

Carotid endarterectomy (CEA) and more recently carotid artery stenting are the treatments of choice for atherosclerotic disease of the extracranial carotid arteries; however, early restenosis caused by neointimal hyperplasia confounds surgical therapy. Oxidative stress has been implicated in the progression of intimal hyperplasia. The authors hypothesized that ketorolac tromethamine (Toradol), a nonsteroidal antiinflammatory drug that is a potent cyclooxygenase inhibitor, would decrease oxidative stress and thereby reduce intimal hyperplasia in a rat CEA model. Twenty-nine male Sprague-Dawley rats underwent CEA and were divided into 3 treatment groups as follows: (1) control (placebo), (2) 7.5 mg/kg Toradol, and (3) 10 mg/kg Toradol. Toradol treatment began 2 days before CEA and continued for 2 weeks. Two weeks after endarterectomy, carotid arteries were fixed, harvested, and examined for platelet activity (platelet reactive units), oxidative stress (malondialdehyde and glutathione), and intimal hyperplasia (measured as percentage of luminal stenosis). Platelet activity, malondialdehyde and glutathione, and intimal hyperplasia were all significantly lowered in both 7.5- and 10-mg/kg doses of Toradol versus control. Toradol given daily beginning 2 days before CEA and ending 2 weeks after the procedure was effective at significantly reducing platelet activity, oxidative stress, and intimal hyperplasia development in the rat without any increase in bleeding. Although the mechanism of action of this reduction is not completely understood, one possible explanation may be through the inhibition of reactive oxygen species production.     

Endoluminal arterial injury in plasminogen-deficient mice

BACKGROUND: Vascular remodeling following arterial injury is characterized by an initial inflammatory reaction. Prior experiments using peritoneal inflammatory models have shown that the plasminogen system plays a role in the intensity of the inflammatory response. This study was undertaken to test the hypothesis that an absence of plasminogen would lead to a decrease in vascular remodeling. METHODS: A left carotid artery injury was created with a flexible guidewire in both wild-type [Plg(+/+)] and plasminogen deficient [Plg(-/-)] mice. The right carotid artery was uninjured and used as a control. Three weeks postinjury, the mice were sacrificed and perfusion fixed, and the bilateral carotid arteries were sectioned for histological examination and collection of morphometric data. RESULTS: After arterial injury, electron microscopy of the acutely injured artery revealed that the endothelium was denuded, that there were breaks in the internal elastic membrane, and that there was disruption of the medial layer of smooth muscle cells. The intimal and medial areas were significantly increased between the uninjured and injured carotid arteries of both Plg(+/+) (+80% intimal, +41% medial, P < 0. 05) and Plg(-/-) [+48% intimal, +24% medial, P < 0.05) mice. However, although there was a significant increase in the adventitial area of Plg(+/+) mice (+18%, P < 0.05), there was no difference in Plg(-/-) mice (-6%). Interestingly, even after 3 weeks, four of six injured arteries in Plg(-/-) mice had persistent thrombus within the medial layer, whereas this was not found in any of the nine Plg(+/+) mouse arteries. DISCUSSION: Plasminogen deficiency inhibited the increase in adventitial area seen after injury in Plg(+/+) mice, but not the increase in intimal or medial areas. Not surprisingly, plasminogen-deficient mice also demonstrated a severe alteration in intramural thrombus clearance. Thus, specific aspects of the vascular remodeling response are dependent on plasminogen.     

Carotid artery anastomosis with albumin solder and near infrared lasers: a comparative study.

BACKGROUND AND OBJECTIVE: Laser tissue-welding has been used for anastomosis of carotid arteries. During welding, thermal injury sustained by the vessel walls should be minimized to prevent thrombosis. Two different types of lasers were used and effects on tissue damage were studied in vitro and in vivo. STUDY DESIGN/MATERIALS AND METHODS: End-to-end anastomosis of dog carotid arteries (n = 10) was performed by using a human albumin solder (HAS) in conjunction with Nd:YAG or diode lasers (lambda = 1.32 microm and 1.9 microm, respectively). The arteries were evaluated for patency and evidence of histologic injury after 21 days. Another group of arteries was laser soldered in vitro to measure the intimal and adventitial temperatures by using thermocouples. RESULTS: The arteries repaired with the diode laser sustained significantly less thermal damage than those repaired with Nd:YAG laser, both in vitro and in vivo. In particular, the intimal temperature was significantly lower (P < 0.05) for the diode than for the Nd:YAG repairs (approximately 35 degrees C and approximately 50 degrees C, respectively). In the latter group, the patency rate was 75%, but thrombosis occurred in 75% of the specimens at 21 days. All diode anastomoses were patent and thrombosis developed in only 17% of the arteries. CONCLUSION: Use of the diode laser and albumin solders may provide a means to successfully repair carotid arteries with minimal thermal damage.     

Recombinant human thrombomodulin inhibits arterial neointimal hyperplasia after balloon injury

OBJECTIVE: Smooth muscle cell proliferation is a major pathophysiologic factor in injury-induced neointimal hyperplasia and recurrent stenosis. We have demonstrated that recombinant human thrombomodulin (rTM) inhibits thrombin-induced arterial smooth muscle cell proliferation in vitro. The purpose of this study was to investigate the effect of rTM on neointimal hyperplasia in vivo. METHODS: A rabbit femoral artery balloon injury model was used. Bilateral superficial femoral arteries were deendothelialized with a 2F arterial embolectomy catheter. rTM (145 microg/kg; 2.0 microg/mL in circulation) or Tris-hydrochloride vehicle control was administered intravenously during the procedure, then either discontinued (group A) or administered twice daily for an additional 48 hours (group B). Rabbits were euthanized at 4 days and at 1, 2, and 4 weeks, and femoral artery specimens were prepared with in situ perfusion fixation and paraffin embedding. Luminal, intima, media, and whole artery areas were quantitated with digital imaging computerized planimetry. Intima-media and lumen-whole artery ratios were calculated. The injury-induced inflammatory reaction was also evaluated with light microscopy, scanning and transmission electron microscopy, and immunohistochemical and immunohistofluorescence staining. RESULTS: In the buffer control group, neointimal hyperplasia after femoral artery balloon injury was evident at 2 weeks, and was pronounced at 4 weeks (P <.0001). Infusion of rTM significantly inhibited intimal hyperplasia at both 2 and 4 weeks (P <.0001). In group A, rTM reduced the intima-media ratio by 27% and 39% at 2 and 4 weeks, respectively. Extended administration of rTM (group B) resulted in inhibition of hyperplasia by 57% and 30% at 2 and 4 weeks, respectively, but failed to reach significance compared with the shorter exposure. rTM infusion significantly inhibited thrombosis (8.1-fold) compared with the buffer control group (P =.012). rTM had no significant effect on lumen area or lumen-whole artery ratio, but treated arteries demonstrated significantly less compensatory dilatation (P =.045), as measured by whole artery area in response to less intimal hyperplasia. rTM administration inhibited platelet adhesion and inhibition of neutrophil infiltration to a degree that approached statistical significance (P =.0675). CONCLUSIONS: Systemic intravenous administration of rTM significantly decreases neointimal hyperplasia and improves patency in the rabbit femoral artery after balloon injury. In addition to exhibiting antithrombotic and antiproliferative effects, rTM may also invoke an anti-inflammatory mechanism, and may alter vascular remodeling in a multidimensional role to inhibit recurrent stenosis after arterial injury.     

The natural history of intimal flaps in a canine model

The natural history of arterial intimal flaps has not been well defined. This study characterizes the natural history of unrepaired intimal flaps. Thirty-nine 1-, 2-, and 3-mm hemispheric, distally based intimal flaps were made in 4- to 5-mm diameter canine femoral and carotid arteries. Twenty arteries had 2- and 3-mm intimal flaps and were monitored for short-term arterial thrombosis and flap extension. Nineteen had 1- and 2-mm intimal flaps and were monitored for thrombosis, long-term development of neointimal hyperplasia, arterial stenosis, and persistence of the flap. While 40% of the arteries with 3-mm intimal flaps developed thrombosis in 3 to 5 days, only 3% of the arteries with 1- or 2-mm intimal flaps developed thrombosis. Most 1- to 2-mm intimal flaps resolved and the subsequent development of neointimal hyperplasia or arterial stenosis was minimal. Arteries with hemodynamically significant stenoses from intimal flaps warrant repair, while arteries with smaller intimal flaps may not require repair.