John Lowery

Intracranial Clot Lysis With Intravenous Microbubbles and Transcranial Ultrasound in Swine

Background and Purpose— Destruction of microbubbles by transcutaneous low-frequency ultrasound (LFUS) has been used to lyse adjacent clot and recanalize acutely thrombosed vessels. LFUS with intraarterial microbubbles has been shown to lyse cerebral clot rapidly in pigs without thrombolytic drugs. We hypothesized that intravenous platelet-targeted microbubbles with LFUS may be a rapid noninvasive technique to recanalize thrombosed intracerebral vessels. Methods— After angiography, 0.5 cc of autogenous thrombus was injected into 1 ascending pharyngeal artery of a pig, occluding it and the rete mirabile. These vessels connect the carotid to the internal carotid and are the main cerebral blood supply. Saline control or intravenous decafluorobutane-sonicated dextrose albumin microbubbles tagged with a subtherapeutic quantity of glycoprotein 2b/3a receptor inhibitor eptifibatide (75 U/kg plus 12 cc of microbubbles administered over 21 minutes), or eptifibatide control, was given with transcutaneous temporal LFUS (1 MHz at 2.0 W/cm2) for 24 minutes. Angiography followed with scoring of declotting and flow. The same protocol was repeated on the contralateral side with the other test fluid so each animal received a saline control and either tagged microbubble or eptifibatide alone. Results— Fifteen pigs completed the protocol with a mean clot age of 4.6 hours. Using tagged microbubbles, 6 of 8 achieved success compared with 0 of 7 receiving eptifibatide alone (P=0.007) and 1 of 15 receiving saline alone (P=0.02). Conclusions— Intravenous platelet-targeted microbubbles combined with transcranial LFUS can rapidly open acute intracranial thrombotic occlusions. Further development for ischemic stroke therapy is justified.

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.

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.

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.

Variations in the Circle of Willis in the New Zealand White Rabbit

PURPOSE The New Zealand White rabbit (NZWR) serves an important role as an experimental model for vascular research, specifically in the area of stroke. Here the authors document vascular variations in the circle of Willis (COW). MATERIALS AND METHODS Subselective internal carotid digital subtraction angiography was performed in 100 NZWRs. RESULTS Important variations include hypoplasia in 36%, duplication of the middle cerebral artery in 29%, asymmetries of the posterior region in 19%, and multiple variations in 28%. The complete classical symmetric COW without significant variation is present in fewer than 30% of animals. CONCLUSIONS With recognition of the variations, the NZWR becomes an improved research model.

Neurological Assessment Scores in Rabbit Embolic Stroke Models

Background: Neurological outcomes and behavioral assessments are widely used in animal models of stroke, but assessments in rabbit models are not fully validated. The wryneck model of neurological assessment scores (NAS) was compared to percent infarct volume (%IV) values (infarct volume is a proven clinical indicator of stroke severity) and arterial occlusion localization in three rabbit angiographic stroke models. Hypothesis: NAS values will correlate with percent infarct volume values. Methods: Anesthetized New Zealand White rabbits (N=131, 4-5 kg) received internal carotid artery emboli by angiographic catheter introduced into the femoral artery and occlusions were characterized. Rabbits were evaluated at 24 hours post embolism using the NAS test of 0 (normal) to 10 (death). Deficit criteria included neck twist, righting reflex, extension reflex in hind paw and forepaw, and posture. Brain sections stained with triphenyltetrazolium chloride (TTC) were analyzed for %IV. Volume of the infarct was measured and calculated as a percent of the total brain volume. Results: The aggregate correlation for NAS values vs. %IV values was R=0.61, p<0.0001, a strong positive relationship, while correlations of the NAS components ranged from R=0.28-0.46. Occlusionsof the posterior cerebral artery vs. the middle cerebral artery alone produced significantly greater deficit scores at p<0.0001. Conclusions: These positive results validate the NAS system in the rabbit angiographic embolic stroke model.

Decreased Serum Levels of S-100B Protein Reflect Successful Treatment Effects in a Rabbit Model of Acute Ischemic Stroke

Serum levels of S-100B were investigated as a marker for infarct volume and response to treatment following acute ischemic stroke in rabbits. Following subselective angiography, rabbits (n=31) were embolized by injection of a 3-day-old blood clot (0.6x4.0-mm) into the internal carotid artery. Treatment began 1-hr post-embolization, groups included: Control (n=8, embolization only), tissue plasminogen activator (tPA, n=12, 0.9mg/kg), and perflutren lipid microbubbles with transcranial ultrasound (MB+US, n=11, MB at 0.16mg/kg, US at 1-MHz pulsed-wave, 0.8 W/cm2 for 1-hr). Serum S-100B levels were significantly increased (P<0.01) 24-hours following embolization in control (3.1-fold over baseline) and tPA (2.9-fold) groups, while treatment with MB+US resulted in an attenuated, non-significant (P=0.221) increase (1.6-fold). Twenty-four hour infarct volumes averaged 4.76%±1.16% for controls, 2.25%±0.95% for rabbits treated with tPA (P=0.32 vs. control), and 0.79%±0.99% for rabbits treated with MB+US (P=0.04 vs. control). Twenty-four hour concentrations of S-100B were positively correlated with infarct volume (r=0.59, P=0.0004).

Persistent penumbra in a rabbit stroke model: incidence and histologic characteristics.

Duration and extent of penumbra determine the window and brain volume in which interventions may save injured tissue after stroke. Understanding the penumbra in animals is necessary in order to design models that translate to effective clinical therapies. New Zealand white rabbits were embolized with aged autologous clot (n = 23) or insoluble microspheres (n = 21). To examine effects of treatment on penumbra, sphere-stroked animals were treated with 3 μm microbubbles plus ultrasound (n = 19). Rabbits were euthanized at 4 or 24 hr. Infarct volume was measured following triphenyltetrazolium chloride (TTC) staining of brain sections. Penumbra was visualized using immunostaining of pimonidazole injected fifteen minutes prior to euthanasia. Potentially reversible penumbra was present in 14.3% stroked rabbits at 4 hours and 15.7% at 24 hours after embolic stroke and represented up to 35% of total lost tissue. Intervention at up to 24 hours may benefit a significant patient population.