Rene Flores

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.

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.

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.

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.