Aliza T. Brown

Successful Microbubble Sonothrombolysis Without Tissue-Type Plasminogen Activator in a Rabbit Model of Acute Ischemic Stroke

Background and Purpose— Microbubbles (MB) combined with ultrasound (US) have been shown to lyse clots without tissue-type plasminogen activator (tPA) both in vitro and in vivo. We evaluated sonothrombolysis with 3 types of MB using a rabbit embolic stroke model. Methods— New Zealand White rabbits (n=74) received internal carotid angiographic embolization of single 3-day-old cylindrical clots (0.6×4.0 mm). Groups included: (1) control (n=11) embolized without treatment; (2) tPA (n=20); (3) tPA+US (n=10); (4) perflutren lipid MB+US (n=16); (5) albumin 3 &mgr;m MB+US (n=8); and (6) tagged albumin 3 &mgr;m MB+US (n=9). Treatment began 1 hour postembolization. Ultrasound was pulsed-wave (1 MHz; 0.8 W/cm2) for 1 hour; rabbits with tPA received intravenous tPA (0.9 mg/kg) over 1 hour. Lipid MB dose was intravenous (0.16 mg/kg) over 30 minutes. Dosage of 3 &mgr;m MB was 5×109 MB intravenously alone or tagged with eptifibatide and fibrin antibody over 30 minutes. Rabbits were euthanized at 24 hours. Infarct volume was determined using vital stains on brain sections. Hemorrhage was evaluated on hematoxylin and eosin sections. Results— Infarct volume percent was lower for rabbits treated with lipid MB+US (1.0%±0.6%; P=0.013), 3 &mgr;m MB+US (0.7%±0.9%; P=0.018), and tagged 3 &mgr;m MB+US (0.8%±0.8%; P=0.019) compared with controls (3.5%±0.8%). The 3 MB types collectively had lower infarct volumes (P=0.0043) than controls. Infarct volume averaged 2.2%±0.6% and 1.7%±0.8% for rabbits treated with tPA alone and tPA+US, respectively (P=nonsignificant). Conclusions— Sonothrombolysis without tPA using these MB is effective in decreasing infarct volumes. Study of human application and further MB technique development are justified.

Microbubbles Improve Sonothrombolysis In Vitro and Decrease Hemorrhage In Vivo in a Rabbit Stroke Model

Introduction:Tissue plasminogen activator (tPA) is the thrombolytic standard of care for acute ischemic stroke, but intracerebral hemorrhage (ICH) remains a common and devastating complication. We investigated using ultrasound (US) and microbubble (MB) techniques to reduce required tPA doses and to decrease ICH. Materials and Methods:Fresh blood clots (3–5 hours) were exposed in vitro to tPA (0.02 or 0.1 mg/mL) plus pulsed 1 MHz US (0.1 W/cm2), with or without 1.12 × 108/mL MBs (Definity or albumin/dextrose MBs [adMB]). Clot mass loss was measured to quantify thrombolysis. New Zealand white rabbits (n = 120) received one 3- to 5-hour clot angiographically delivered into the internal carotid artery. All had transcutaneous pulsed 1 MHz US (0.8 W/cm2) for 60 minutes and intravenous tPA (0.1–0.9 mg/kg) with or without Definity MBs (0.16 mL/mg/kg). After killing the animals, the brains were removed for histology 24 hours later. Results:In vitro, MBs (Definity or adMB) increased US-induced clot loss significantly, with or without tPA (P < 0.0001). At 0 and 0.02 mg/mL, tPA clot loss was greater with adMBs compared with Definity (P ≤ 0.05). With MB, the tPA dose was reduced 5-fold with good efficacy. In vivo, both Definity MB and tPA groups had less infarct volume compared with controls at P < 0.0183 and P = 0.0003, respectively. Definity MB+tPA reduces infarct volume compared with controls (P < 0.0001), and ICH incidence outside of strokes was significantly lower (P = 0.005) compared with no MB. However, infarct volume in Definity MB versus tPA was not different at P = 0.19. Conclusion:Combining tPAand MB yielded effective loss of clot with very low dose or even no dose tPA, and infarct volumes and ICH were reduced in acute strokes in rabbits. The ability of MBs to reduce tPA requirements may lead to lower rates of hemorrhage in human stroke treatment.

Microbubble Augmented Ultrasound Sonothrombolysis Decreases Intracranial Hemorrhage in a Rabbit Model of Acute Ischemic Stroke

Objectives:Increasing evidence confirms that microbubble (MB)-augmented ultrasound (US) thrombolysis enhances clot lysis with or without tissue plasminogen activator (tPA). Intracranial hemorrhage (ICH) is a major complication militating against tPA use in acute ischemic stroke. We quantified the incidence of ICH associated with tPA thrombolysis and MB + US therapy and compared infarct volumes in a rabbit model of acute ischemic stroke. Materials and Methods:Rabbits (n = 158) received a 1.0-mm clot, angiographically injected into the internal carotid artery causing infarcts. Rabbits were randomized to 6 test groups including (1) control (n = 50), embolized without therapy, (2) US (n = 18), (3) tPA only (n = 27), (4) tPA + US (n = 22), (5) MB + US (n = 27), and (6) tPA + MB + US (n = 14). US groups received pulsed wave US (1 MHz, 0.8 W/cm2) for 1 hour; rabbits with tPA received intravenous tPA (0.9 mg/kg) over 1 hour. Rabbits with MB received intravenous MB (0.16 mg/kg) given over 30 minutes. Rabbits were killed 24 hours later and infarct volume and incidence, location, and severity of ICH were determined by histology and pathologic examination. Results:Percentage of rabbits having ICH outside the infarct area was significantly decreased (P = 0.004) for MB + US (19%) rabbits compared with tPA + US (73%), US only (56%), tPA (48%), tPA + MB + US (36%), and control (36%) rabbits. Incidence and severity of ICH within the infarct did not differ (P > 0.39). Infarct volume was significantly greater (P = 0.002) for rabbits receiving US (0.97% ± 0.17%) than for MB + US (0.20% ± 0.14%), tPA + US (0.15% ± 0.16%), tPA (0.14% ± 0.14%), and tPA + MB + US (0.10% ± 20%) rabbits; these treatments collectively, excluding US only, differed (P = 0.03) from control (0.45% ± 0.10%). Conclusions:Treatment with MB + US after embolization decreased the incidence of ICH and efficacy was similar to tPA in reducing infarct volume.

Homocysteine-induced vascular dysregulation is mediated by the NMDA receptor

Elevated plasma homocysteine accelerates myointimal hyperplasia and luminal narrowing after carotid endarterectomy. N-methyl D aspartate receptors (NMDAr) in rat cerebrovascular cells are involved in homocysteine uptake and receptor-mediated stimulation. In the vasculature, NMDAr subunits (NR1, 2A-2D) have been identified by sequence homology in rat aortic endothelial cells. Exposure of these cells to homocysteine increased expression of receptor subunits, an effect that was attenuated by dizocilpine (MK801), a noncompetitive NMDA inhibitor. The objective of this study was to investigate the existence of an NMDAr in rat vascular smooth muscle (A7r5) cells, and also the effect of homocysteine on vascular dysregulation as mediated by this receptor. Subunits of the NMDAr (NR1, 2A-2D) were detected in the A7r5 cells by using the reverse transcriptase polymerase chain reaction and Western blotting. Homocysteine induced an increase in A7r5 cell proliferation, which was blocked by MK801. Homocysteine, in a dose and time dependent manner, increased expression of matrix metallinoproteinase-9 and interleukin-1beta, which have been implicated in vascular smooth muscle cell migration and/or proliferation. Homocysteine reduced the vascular elaboration of nitric oxide and increased the elaboration of the nitric oxide synthase inhibitor, asymmetric dimethylarginine. All of these homocysteine mediated effects were inhibited by MK801. NMDAr exist in vascular smooth muscle cells and appear to mediate, at least in part, homocysteine-induced dysregulation of vascular smooth muscle cell functions.

Increasing levels of dietary homocystine with carotid endarterectomy produced proportionate increases in plasma homocysteine and intimal hyperplasia.

PURPOSE The role that homocysteine may play in post-carotid endarterectomy (CEA) restenosis due to intimal hyperplasia is not well understood. This study was designed to investigate the effects of different levels of dietary homocystine on: (1) plasma homocysteine; (2) post-CEA intimal hyperplasia; and (3) levels of the methyl donor S-adenosylmethionine (SAM) and its counterpart S-adenosylhomocysteine (SAH) in the homocysteine pathway. METHODS Male rats were fed specialized diets for 2 weeks pre- and post-CEA. Groups included control (0 homocystine added, n=9), 1.5 (1.5 g/kg homocystine added, n=10), 3.0 (3.0 g/kg homocystine added, n=9), and 4.5 (4.5 g/kg homocystine added, n=11). The rats underwent a surgical carotid endarterectomy. Endpoints included; plasma homocysteine, intimal hyperplasia, replicative index using with alpha-SM actin and BrdU, hepatic SAM levels, SAH levels, and the hepatic activities of methylenetetrahydrofolate reductase (MTHFR) and cystathionine beta-synthase (CBS). RESULTS Increasing dietary homocystine produced a proportionate increase in plasma homocysteine and an increase in intimal hyperplasia. Regression analysis of plasma homocysteine levels and intimal hyperplasia showed a significant correlation (r=0.71,P=0.003). Plasma homocysteine levels above 15 microM were associated with significant increases in intimal hyperplasia above 6.5% (P=0.04). Elevation of plasma homocysteine levels to moderate levels (5-25 microM) resulted in significant post-CEA intimal hyperplasia. Cellular analysis of the area of intimal hyperplasia in all diet groups showed comparable amounts of cells positive for alpha-SM actin. However, with increasing levels of dietary homocystine and plasma homocysteine there was an increase in replicative index (P<0.001) as determined by BrdU staining. Increasing dietary homocystine increased plasma homocysteine and was followed by increases in the replicative index thus producing increased intimal hyperplasia and lumenal stenosis. In hepatic measurements the 1.5 and 3.0 g/kg homocystine diets caused: increased liver activity of MTHFR (P=0.03) and decreased hepatic levels of SAM, SAH and SAM/SAH ratios compared to controls. Homocystine treatment did not cause significant alterations in CBS levels (P=0.992). These studies also showed no correlation of the MTHFR and CBS enzymes with plasma homocysteine levels or intimal hyperplasia. However, hepatic levels of SAM showed significant negative correlations with plasma homocysteine (r=-0.58; P=0.006) and with BrdU percentages of cellular proliferation (r=-0.69; P=0.06). CONCLUSION The degree of post-CEA intimal hyperplasia in a rat model is directly related to the plasma level of homocysteine. The hyperplastic effects of homocysteine may be mediated in part by a physiological insufficiency of methyl donors as shown by decreases in SAM. Thus, increasing levels of plasma homocysteine enhanced and accelerated the smooth muscle cell response after CEA which led to increased intimal hyperplasia and lumenal stenosis.

Saratin, an inhibitor of collagen-platelet interaction, decreases venous anastomotic intimal hyperplasia in a canine dialysis access model.

Prosthetic dialysis access thrombosis and/or stenosis is the most common cause of graft impairment or loss and is primarily attributed to venous outflow stenosis due to intimal hyperplasia. Intimal hyperplasia is thought to result from interactions between areas of exposed subendothelial collagen in an injured vessel and platelets, resulting in platelet adhesion. Saratin, an inhibitor of the WF-dependent binding of platelet to collagen interaction, has been shown in vitro to reduce the adhesion of platelets to collagen. In the current study, the authors inves-tigated the effects of topical saratin administration in a canine dialysis access model in regard to intimal hyperplasia development at the venous anastomosis. Fourteen female mongrel dogs underwent placement of a femoral polytetrafluoroethylene (PTFE) dialysis access graft and were placed into 1 of 2 groups: 1) control or 2) experimental with topical saratin application. The experimental group had 600,μg of saratin (1 μg/,μL) applied for 5 minutes directly onto the venous anastomosis before restoration of blood flow; control groups received vehicle control. At 4 weeks postoperative, a portion of the graft was removed along with a segment of the outflow vein. Veins were subsequently processed, sectioned, and analyzed along the length of the excised segment and divided into blocks that included the area of the graft toe, midanastomotic region and heel, and blocks A-E. Intimal hyperplasia was assessed by a computerassisted morphometric analysis. Platelet counts and bleeding times were also measured. Vein segments in the control group (n= 7) showed pronounced intimal hyperplasia in blocks B, C, and D as compared to the saratin group (n = 6). Distribution of intimal hyperplasia by blocks between control and saratin groups were as follows: block [A] 8.6 ±1.9 vs 9.7 ±3.0% (p = NS), [B] 32.7 ±6.3 vs 10.7 ±3.5% (p=0.01), [C] 44.8 ±6.2% vs 10.3 ±1.5% (p=0.0004), [DI 40.8 ±1 1.0 vs 9.1 ±4.2% (p = 0.02), [E] 7.5 ±5.5 vs 2.7 ±0.4% (p = NS). Intimal hyperplasia normalized to vein wall thickness also showed a significant reduction with saratin application. Bleeding times and platelet counts obtained at different time points during the experiment showed no difference between control and saratin groups. In a canine dialysis access model using PTFE grafts, topical application of saratin at the venous anastomosis decreased intimal hyperplasia development by as much as 77% when compared with control animals. Saratin provides for a method of substantially reducing intimal hyperplasia by direct local application without systemic side effects.

Dodecafluoropentane Emulsion Decreases Infarct Volume in a Rabbit Ischemic Stroke Model

PURPOSE To assess the efficacy of dodecafluoropentane emulsion (DDFPe), a nanodroplet emulsion with significant oxygen transport potential, in decreasing infarct volume in an insoluble-emboli rabbit stroke model. MATERIALS AND METHODS New Zealand White rabbits (N = 64; weight, 5.1 ± 0.50 kg) underwent angiography and received embolic spheres in occluded internal carotid artery branches. Rabbits were randomly assigned to groups in 4-hour and 7-hour studies. Four-hour groups included control (n = 7, embolized without treatment) and DDFPe treatment 30 minutes before stroke (n = 7), at stroke onset (n = 8), and 30 minutes (n = 5), 1 hour (n = 7), 2 hours (n = 5), or 3 hours after stroke (n = 6). Seven-hour groups included control (n = 6) and DDFPe at 1 hour (n = 8) and 6 hours after stroke (n = 5). DDFPe dose was a 2% weight/volume intravenous injection (0.6 mL/kg) repeated every 90 minutes as time allowed. After euthanasia, infarct volume was determined by vital stains on brain sections. RESULTS At 4 hours, median infarct volume decreased for all DDFPe treatment times (pretreatment, 0.30% [P = .004]; onset, 0.20% [P = .004]; 30 min, 0.35% [P = .009]; 1 h, 0.30% [P = .01]; 2 h, 0.40% [P = .009]; and 3 h, 0.25% [P = .003]) compared with controls (3.20%). At 7 hours, median infarct volume decreased with treatment at 1 hour (0.25%; P = .007) but not at 6 hours (1.4%; P = .49) compared with controls (2.2%). CONCLUSIONS Intravenous DDFPe in an animal model decreases infarct volumes and protects brain tissue from ischemia, justifying further investigation.

Stroke Location and Brain Function in an Embolic Rabbit Stroke Model

PURPOSE Current rabbit stroke models often depend on symptoms as endpoints for embolization and produce wide variation in location, size, and severity of strokes. In a further refinement of an angiographic embolic stroke model, localized infarctions were correlated to neurologic deficits with the goal to create a rabbit model for long-term studies of therapies after stroke. MATERIALS AND METHODS New Zealand White rabbits (4-5 kg; N = 71) had selective internal carotid artery (ICA) angiography and a single clot was injected. At 24 hours, neurologic assessment score (NAS) was measured on an 11-point scale (0, normal; 10, dead). Brains were removed and stained to identify stroke areas. All animals with single strokes (n = 31) were analyzed by specific brain structure involvement, and NAS values were correlated. RESULTS Stroke incidence differed by location, with cortex, subcortical, and basal ganglia regions highest. The middle cerebral artery (MCA), at 52%, and anterior cerebral artery (ACA), at 29%, were most commonly involved, with the largest stroke volumes in the ACA distribution. Brainstem and cerebellum strokes had disproportionately severe neurologic deficits, scoring 2.25 +/- 1.0 on the NAS, which represented a significant (P < .02) difference versus cortex (0.5 +/- 0.2), subcortical (1.3 +/- 0.4), and basal ganglia (0.5 +/- 0.3), all in the frontal or parietal regions. CONCLUSIONS MCA and ACA distributions included 81% of strokes. These sites were relatively silent (potentially allowing longer-term survival studies) whereas others in the posterior circulation produced disproportionately severe symptoms. Symptoms were not reliable indicators of stroke occurrence, and other endpoints such as imaging may be required. These are important steps toward refinement of the rabbit stroke model.

Three variations in rabbit angiographic stroke models.

PURPOSE To develop angiographic models of embolic stroke in the rabbit using pre-formed clot or microspheres to model clinical situations ranging from transient ischemic events to severe ischemic stroke. MATERIALS AND METHODS New Zealand White rabbits (N=151) received angiographic access to the internal carotid artery (ICA) from a femoral approach. Variations of emboli type and quantity of emboli were tested by injection into the ICA. These included fresh clots (1.0-mm length, 3-6h), larger aged clots (4.0-mm length, 3 days), and 2 or 3 insoluble microspheres (700-900 μm). Neurological assessment scores (NAS) were based on motor, sensory, balance, and reflex measures. Rabbits were euthanized at 4, 7, or 24h after embolization, and infarct volume was measured as a percent of total brain volume using 2,3,5-triphenyltetrazolium chloride (TTC). RESULTS Infarct volume percent at 24 h after stroke was lower for rabbits embolized with fresh clot (0.45±0.14%), compared with aged clot (3.52±1.31%) and insoluble microspheres (3.39±1.04%). Overall NAS (including posterior vessel occlusions) were positively correlated to infarct volume percent measurements in the fresh clot (r=0.50), aged clot (r=0.65) and microsphere (r=0.62) models (p<0.001). CONCLUSION The three basic angiographic stroke models may be similar to human transient ischemic attacks (TIA) (fresh clot), major strokes that can be thrombolysed (aged clot), or major strokes with insoluble emboli such as atheromata (microspheres). Model selection can be tailored to specific research needs.

Effect of clopidogrel on platelet aggregation and intimal hyperplasia following carotid endarterectomy in the rat.

Intimal hyperplasia results in significant morbidity and mortality following vascular intervention. Both platelets and elevated homocysteine have been implicated in the development of intimal hyperplasia. We previously demonstrated that a locally applied antiplatelet agent decreases the development of intimal hyperplasia. We were therefore interested in a systemic antiplatelet agent, clopidogrel. We hypothesized that clopidogrel would decrease platelet aggregation and activity and intimal hyperplasia. Male Sprague-Dawley rats underwent carotid endarterectomy (CEA) and treatment with either placebo or varying regimens of clopidogrel, including chronic, pre-CEA bolus, chronic plus pre-CEA bolus, and chronic plus post-CEA bolus; a homocystine diet was used to elevate both plasma homocysteine and the degree of intimal hyperplasia. Platelet aggregation, platelet activity, and intimal hyperplasia were then assessed. Platelet aggregation was not decreased with chronic clopidogrel; however, it was decreased with pre-CEA bolus clopidogrel. Similarly, platelet activity was not inhibited by chronic clopidogrel but was inhibited by pre-CEA and chronic plus pre-CEA bolus clopidogrel. Neither chronic, pre-CEA bolus, chronic plus pre-CEA bolus, nor chronic plus post-CEA bolus clopidogrel resulted in a decrease in intimal hyperplasia. Although pre-CEA bolus clopidogrel resulted in a decrease in both platelet aggregation and activity, it was unable to decrease the development of intimal hyperplasia at any dose. Additional factors must therefore contribute to the pathologic development of intimal hyperplasia.