An Echogenic Clot Method for Thrombolysis Monitoring in Thrombotic Stroke Models

To demonstrate thrombolytic efficacy of a tissue plasminogen activator (tPA)-loaded echogenic liposome (TELIP) formulation in a rabbit thrombotic stroke model (the most relevant animal model for evaluation of directed thrombolytic therapy for ischemic stroke), we sought to develop a means of monitoring thrombus dissolution quantitatively by ultrasound imaging methods. We hypothesized that a gas-free ultrasound contrast agent can be incorporated into blood clots at a concentration that does not affect the tPA-mediated clot dissolution rate, while enabling quantitative assessment of the clot dissolution rate. Clots were formed from a mixture of whole rabbit blood, 1 M calcium chloride, human thrombin and varying amounts of microcrystalline cellulose. Washed clots in tubes were weighed at 30, 60 and 90 minutes after addition of recombinant tPA (rtPA) in porcine plasma (100 μg/ml). Clot echogenicity at each time point was assessed using a Philips HDI 5000 ultrasound system using an L12-5 linear array probe. Recorded Images underwent videodensitometric analysis that converted image reflectivity to mean gray scale values (MGSV). We found that 1.12 mg/ml of microcrystalline cellulose in rabbit blood clots (0.2 ml) provided optimal echogenicity without affecting clot dissolution rates (0.3-0.6 mg/min.) caused by rtPA. The clot dissolution rate measured by videodensitometric analysis of the echogenic clots agreed well with that determined by mass loss measurements (0.28% 0-time value/minute). This method will be important for demonstrating in vivo efficacy with potentially decreased hemorrhagic effects provided by directed tPA vehicles relative to systemic administration of the free thrombolytic.