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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 μm MB+US (n=8); and (6) tagged albumin 3 μm MB+US (n=9). Treatment began 1 hour postembolization. Ultrasound was pulsed-wave (1 MHz; 0.8 W/cm 2 ) 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 μm MB was 5×10 9 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 μm MB+US (0.7%±0.9%; P =0.018), and tagged 3 μm 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.

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.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.In vitro, MBs (Definity or adMB) increased US-induced clot loss significantly, with or without tPA (P0.0001). 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 P0.0183 and P = 0.0003, respectively. Definity MB+tPA reduces infarct volume compared with controls (P0.0001), 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.Combining tPA and 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.

Introduction: Neuroprotective strategies in ischemic stroke include oxygen delivery to sustain penumbra and prevent hypoxic cell death. Hyperbaric oxygen, blood substitutes, and liquid fluorocarbon-based oxygen carriers have often failed in treatment of stroke and other ischemic disorders. Dodecafluoropentane emulsion (DDFPe, boiling point 29°C) shifts to quasi-gas phase at body temperature, which allows absorption and transportation of very high levels of oxygen. Exceptionally small particle size, 250-300 nm, may allow oxygen delivery even through occluded vessels, by diffusion into hypoxic tissue unreachable by whole blood. In a preliminary stroke study in rabbits, DDFPe reduced infarct volumes in all experimental groups by 80% or more. Hypothesis: Repeated doses of DDFPe can reduce infarct volume for up to 24 hours after permanent cerebral artery occlusion in rabbits. Methods: New Zealand White rabbits (N=55) received cerebral angiography from a femoral artery approach. Embolic microspheres (diameter=700-900 μm) were injected into the internal carotid artery, permanently occluding the middle cerebral and/or anterior cerebral arteries. Rabbits were randomly assigned to treatment groups and sacrifice times as in Table 1. In all treated groups, intravenous DDFPe dosing with a 2% w/v emulsion began at 1 hour post-embolization and was repeated every 90 minutes until sacrifice at either 7 or 24 hours post-embolization. Following sacrifice, infarcts were measured as a percent of brain volume using vital stains on brain sections. Results: Percent infarct volume means significantly decreased for all DDFPe treated groups compared with controls (Table 1). Conclusion: Intravenous DDFPe begun 1 hour after stroke onset protects the brain from ischemic injury in the rabbit model of permanent embolic stroke. Decreased infarct volumes represent salvaged brain tissue. This effect can be observed for 24 hours with repeated doses.

Introduction: Intravenous dodecafluoropentane emulsion (DDFPe) has proven ability to transport oxygen in ischemic and anemic animal models. Therapeutic effects in rabbits and pigs last at least 90 minutes, although in human studies dodecafluoropentane (DDFP) is eliminated rapidly from the blood through respiration. DDFPe neuroprotection reduces infarct volumes >80% in our rabbit embolic stroke model. Since little is known about DDFPe activity in rabbits, blood pharmacokinetics were investigated in healthy rabbits to determine dosing regimens. Hypothesis: We hypothesize that DDFP pharmacokinetics in the rabbit are similar to those in humans, with blood half-life values near 2.2±1.2 min, and clearance values near 49.6±10.8 mL/min/kg. Methods: Bilateral ear catheters, one intravenous and one intraarterial, were introduced into New Zealand White rabbits (N=8, 4.2±0.9 kg) for DDFPe administration and blood sampling, respectively. Rabbits were divided into 3 DDFPe dosage groups: 1) single bolus (0.6 mL/kg) with blood sampling for 4 hrs (N=4), 2) 15 doses (0.1 mL/kg) with sampling for 24 hrs (N=2), 3) 5 doses (0.6 mL/kg) with sampling for 7 hrs (N=2). Repeated doses of 2% w/v DDFPe were administered every 90 min. All samples were kept frozen until thawed before analysis by headspace gas chromatography-mass spectrometry. Results: An exponential regression closely approximated the blood concentration vs. time plot of single bolus sampling with an average R 2 of 0.96±0.02, thus compartmental modeling was used for pharmacokinetic analysis. The blood half-life was 1.9±0.6 min, with a clearance of 90.1±45.3 mL/min/kg. In all multi-dose rabbits, blood concentration of DDFP did not reach a steady state. The concentration vs. time data exhibited a repeated sequence of individual bolus data, with DDFP reaching undetectable levels 10 min after each dose. Conclusion: Blood elimination half-life and clearance rates of DDFPe in rabbits are comparable to humans, further indicating that the rabbit stroke model is appropriate for investigation of the neuroprotective potential of DDFPe. The time disparity between blood levels and therapeutic oxygen transport ability of DDFPe suggests multiple compartments and merits further investigation.

Sonothrombolysis (STBL) uses ultrasound to lyse thrombi that cause stroke, myocardial infarct and other vascular occlusion diseases; and efficacy is improved markedly by combining tPA and/or microbubbles (MBs) with ultrasound. The efficacy of in vitro STBL varies considerably depending upon how blood clots are formed. However, the relationship between the physical and chemical nature of blood clots and STBL efficacy has not been quantified. This study measured the effects that clot age, density and thrombin content had on STBL efficacy and the ability of tPA and MBs to complement STBL. Blood clots with different characteristics were produced by mixing pooled rabbit plasma with rabbit blood cells, adding varied amounts of thrombin and calcium, followed by incubation at 37 o C in glass tubes for 3 h to 72 h. Blood clots were then cured at 5 o C for 24 h to 72 h. The volume and mass density of the clots were measured, 9-11 mg pieces were cut from the main clot, weighed and then insonated with 1 MHz, pulsed (20% duty factor) ultrasound in a Mylar chamber through which fresh rabbit serum containing tPA and or MBs was flowed continuously past the clot. STBL efficacy was measured as % of clot mass lysed in 15 min. For clots incubated 3 h to 24 h at 37 o C with 5 mM Ca + 2 and 5 U of thrombin, maximal STBL with ultrasound plus tPA was observed with 0.1 mg/ml tPA for all ultrasonic intensities tested. Using MBs (1.0-2.5 x10 8 /ml) produced a comparable level of STBL. More effective STBL was produced by combining tPA with MBs, even with reduced tPA concentrations (0.006-0.01 mg/ml). However, increasing clot thrombin, up to 60 U, proportionally reduced STBL efficacy with tPA or MBs, and combining tPA with MBs no longer produced an additive STBL effect. This was also true for clots incubated with 25 mM Ca + 2 that did not contract and thus had a fourfold lower mass density. Increasing thrombin levels produced clots whose STBL efficacy was markedly lower with either tPA or MBs, and for which combining tPA with MBs produced no additive STBL effect. Hence, the level of thrombin used during clot formation (and putatively the degree of fibrin networks) was the major factor in determined STBL efficacy and whether or not tPA combined with MBs were additive in potentiating STBL. Surprisingly, the mass density of the clot alone had no effect on STBL efficacy.