RESEARCH AREAS

Combined ultrasound and tissue plasminogen activator (rt-PA) therapy, or sonothrombolysis, has been shown to improve recanalization in patients with acute ischemic stroke. Effective methods of enhancing thrombolysis have been examined in an attempt to reduce the dosage of the thrombolytic agent and reduce the risk of hemorrhagic events. We have investigated the synergistic effect of rt-PA and 120-KHz ultrasound on thrombolysis using in vitro porcine and human whole blood clot models. In our ongoing studies, we have demonstrated that significant enhancement of thrombolysis correlates with the presence of stable cavitation and this type of gentle bubble activity can be sustained using an intermittent infusion of a contrast agent. In addition, we have shown that inertial cavitation, which elicits broadband acoustic emissions, is counter-productive for enhanced thrombolysis. Rather, the most effective form of bubble activity is stable cavitation, which elicits ultrasonic subharmonic generation. These data strongly support the central hypothesis that ultrasound enhances thrombolysis primarily via mechanical mechanisms. Importantly, we have shown encapsulation of rt-PA in a contrast agent specifically targeted to clot. We enlist agents specifically targeted to the thrombus which are echogenic and can be activated by US to improve thrombolysis, allow direct targeting, allow for the reduction of the rt-PA dose, and decrease complications associated with giving this agent systemically. In vitro, and in vivo studies are being performed to test the rt-PA-loaded echogenic liposomes and optimize the ultrasonic technique to maximize US-enhanced thrombolysis. Our long-term objective is to develop a transcranial, ultrasound-enhanced thrombolysis system that minimizes the risk of intracranial hemorrhage, increases the number of stroke survivors, improves long-term prognosis, and reduces health care costs. The development of the agents and techniques in the UC Ultrasound Research Laboratory have far reaching implications in improving directed therapeutic treatment to stroke.

Immunofluorescent staining of mature porcine whole-blood clot.

The clot is located to the left of each panel. Panels A and B are stained with goat-antihuman rt-PA with a secondary of donkey-anti-goat conjugated to FITC. Panels C and D are stained with mouse anti-plasminogen and donkey-anti-mouse conjugated to Rhodamine. Panels A and C are treated with rt-PA in plasma alone and panels B and D were treated with rt-PA in plasma and 120-kHz pulsed ultrasound. These data show that ultrasound treatment dramatically increased rt-PA penetration into the clot.

Echogenic liposomes (ELIP) are being developed at UTHSC-Houston for use as targeted ultrasound contrast agents and drug carriers for ultrasound-triggered drug delivery. Physical and acoustical characterization of ELIP is under way in the UC Ultrasound Research Laboratory to determine the optimum acoustic parameters for diagnostic and therapeutic applications. The utility of ELIP for contrast depends on their stability in an acoustic field, whereas the use of ELIP for drug delivery requires the liberation of encapsulated gas and drug payload at the desired treatment site. Our long-term goals are to determine, quantitate, and characterize the stage, extent, and pathophysiologic development of atherosclerosis, allowing directed therapy to improve physiologic flow following clinical intervention.

Our long-term goal is to develop therapeutic-loaded echogenic liposomes (ELIP) which, when exposed to pulsed ultrasound, trigger drug or gene intravascular release and enhance uptake in targeted vascular beds. Therapeutic agents of interest include an anti-inflammatory (rosiglitazone), an anti-angiogenesis (bevacizumab), and an anti-inflammatory gene (eNOS). Experiments in the UC Ultrasound Research Laboratory are focused on the development of optimal ultrasound parameters for therapeutic delivery and determination of the efficacy of therapeutic release to arrest atheroma progression.

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Screen capture of ELIP circulating in a flow phantom exposed to color Doppler ultrasound from a CL15-7 transducer on a Philips HDI 5000 ultrasound system at an MI of 1.3 (Color Doppler window removed by software).

ELIP rapidly lose echogenicity as they pass through the ultrasound field from right to left. This demonstrates how gas and therapeutic agents may be released from circulating ELIP exposed to ultrasound from a clinical diagnostic scanner.

Echogenic liposomes (ELIP) have been used to entrap micro- and nanobubbles, enabling enhanced echogenicity and cavitation nucleation. The use of ultrasound to fragment drug-loaded ELIP near the target tissue, rather than relying on more gradual passive release, has the potential to produce a large temporal peak in drug or therapeutic effect. This is particularly important at the endothelium where the constant flow of blood may carry away the released drug rapidly, making it unavailable for uptake across the endothelium. Studies are ongoing in the UC Ultrasound Research Laboratory uses a novel ex vivo arterial model to examine delivery of fluorescently-labeled and anti-ICAM-1-targeted ELIP to, and possibly beyond, the endothelium. Our ex vivo test model uses murine wild-type and atheromatous aortae, or porcine normal and atheromatous carotids, and allows quantification of fluorescently labeled and drug-loaded-ELIP in the intravascular and extravascular fluids separately. This platform provides a model system for evaluation of delivery methodologies to the arterial wall.

Ex vivo murine aortas treated with Rhodamine-labeled ELIP delivered via a proximal injection of Rh-ELIP into an intravascular flow of 0.5% BSA at 5.6 mL/min.

A -C show an artery treated with Rh-ELIP alone, while D - F show an artery treated with a combination of Rh-ELIP and CW ultrasound (0.49 MPa peak to peak amplitude). Panels A and D show arteries stained with factor VIII to highlight the endothelium. Panels B and E show the arterial walls as viewed with a blue filter, superimposed over the same segment viewed with a red filter. Panels C and F show the red-filtered image alone, where the increased fluorescence in Panel F is due to the presence of Rhodamine. The full scale bar is 100 µm.

Currently, the only therapy for ischemic stroke (other than aspirin and surgery) that is FDA approved is the thrombolytic agent recombinant tissue Plasminogen Activator (rt-PA). Studies have shown that rt-PA is effective in lysing blood clots in ischemic stroke patients if given within 3 hours after the onset of stroke symptoms. Unfortunately, only about 2 to 7% of ischemic stroke patients actually receive rt-PA due to various factors. One factor is the lack of recognizing and diagnosing strokes in a timely manner. Physicians are also reluctant to administer rt-PA because of the resulting increased risk of intracranial hemorrhage (ICH). Administering rt-PA to a misdiagnosed hemorrhagic stroke patient could be serious and life threatening.

Any adjuvant therapy that lowers the dose of rt-PA or increases its efficacy would represent a significant breakthrough. Improved effectiveness or greater safety would provide a powerful impetus for physicians to administer rt-Pa to a larger portion of the patients with ischemic stroke.

Experimental setup for assessing the combined thrombolytic action of ultrasound and r-tPA:

The custom-designed transducer to the right of the picture exposes a whole blood clot bathing in plasma and rt-PA within the central holder to ultrasound in a temperature-controlled water tank. The clot is then removed and weighed so as to assess percent mass loss relative to its initial mass. The two sound absorvers to the left of the picture are used to prevent the generation of reflections and standing waves during ultrasound exposure.

Recent studies have demonstrated that simultaneous exposure of blood clots to ultrasound and rt-PA results in an increased thrombolytic effect. The Ultrasound Laboratory at the University of Cincinnati is currently developing a novel transcranial ultrasound thrombolysis system (TUTS). With TUTS, an ultrasonic transducer is held against the temporal bone; energy radiates from the transducer, through the bone, and into the brain. It is hoped that application of ultrasound via TUTS will permit the usage of lower dosages of rt-PA than are currently used clinically, yet resulting in increased thrombolysis with reduced risks of hemorrhage. The benefits of the TUTS system potentially include an increased number of stroke survivors, improved long-term prognosis and reduced health care costs.

Recent increases in the pressure output of diagnostic ultrasound scanners have generated concern as to the potentially damaging effects of ultrasound on various tissues. This type of damage may be mediated by the expansion and violent collapse of gas bubbles exposed to ultrasonic excitation, a phenomenon known as inertial cavitation. This has led to an interest in establishing thresholds for bioeffects in many organs, including the lungs of mammals. In order to explore the hypothesis of cavitation-based bioeffects, the Ultrasound Laboratory is carrying out an extensive in vivo investigation of the thresholds of damage in rat lungs exposed to diagnostic ultrasound.

Histopathology of petechial hemorrhage in rat lung exposed to 6 MHz Doppler Pulse for 1.5 minutes at an MI=1.5.

Histopathology of normal rat lung.