Pulsed high-intensity focused ultrasound enhances thrombolysis in an in vitro model

Abstract

Purpose: To evaluate the use of pulsed high-intensity focused ultrasound exposures to improve tissue plasminogen activator (tPA)-mediated thrombolysis in an in vitro model.

Materials and methods: All experimental work was compliant with institutional guidelines and HIPAA. Clots were formed by placing 1 mL of human blood in closed-off sections of pediatric Penrose tubes. Four experimental groups were evaluated: control (nontreated) clots, clots treated with pulsed high-intensity focused ultrasound only, clots treated with tPA only, and clots treated with pulsed high-intensity focused ultrasound plus tPA. The focused ultrasound exposures (real or sham) were followed by incubations of the clots in tPA with saline or in saline only. Thrombolysis was measured as the relative reduction in the mass of the clot. D-Dimer assays also were performed. Two additional experiments were performed and yielded dose-response curves for two exposure parameters: number of pulses per raster point and total acoustic power. Radiation force-induced displacements caused by focused ultrasound exposures were simulated in the clots. A Tukey-Kramer honestly significant difference test was performed for comparisons between all pairs of experimental groups.

Results: The clots treated with focused ultrasound alone did not show significant increases in thrombolysis compared with the control clots. The clots treated with focused ultrasound plus tPA showed a 50% ([30.2/20.1]/20.1) increase in the degree of thrombolysis compared with the clots treated with tPA only (P < .001), further corroborating the d-dimer assay results (P < .001). Additional experiments revealed how increasing both the number of pulses per raster point and the total acoustic power yielded corresponding increases in the thrombolysis rate. In the latter experiment, simulations performed at a range of power settings revealed a direct correlation between increased displacement and observed thrombolysis rate.

Conclusion: The rate of tPA-mediated thrombolysis can be enhanced by using pulsed high-intensity focused ultrasound exposure in vitro.

Figure 1

(a)Dialysis tube closures used to enclose blood samples in latex tubes. Altered closures are shown on the left; arrow points to the region that has been cut away. (b) Two blood samples enclosed by using dialysis closures in the latex tubes. (c) Clots with the enclosed tubes, which are held upright in a tank of degassed water directly opposite the high-intensity focused ultrasound transducer (arrow). A probe containing a digital thermometer used to monitor the tank water temperature is shown on the left.

Figure 2

B-mode ultrasonograms of clots (in latex tubes) lined up for pulsed high-intensity focused ultrasound exposure. Large arrows point to guide marks for the transducer’s focal zone; small arrow points to raster points spaced 2 mm apart. Lateral scan shows one clot. Vertical scan shows two clots, positioned one on top of each other, with the top clot marked for pulsed high-intensity focused ultrasound exposure.

Figure 3

Graph shows results of nine trials performed in the preliminary experiments. (See Table 1 for pulsed high-intensity focused ultrasound [HIFU] exposure and tPA incubation details.) A trend of increasing disparity in the degree of clot size reduction was observed as the pulsed high-intensity focused ultrasound and tPA incubation parameter levels were varied from trials 1-9. Bars represent group means.

Figure 4

Graph shows results of the standard pulsed high-intensity focused ultrasound (HIFU) experiments. The degree of thrombolysis in clots treated with focused ultrasound plus tPA was significantly greater than the degree of thrombolysis in clots treated with tPA only. In both these groups, the degree of thrombolysis was significantly greater than that in the control (untreated) clots and the clots treated with focused ultrasound exposures only. Differing lowercase letters between mean levels of thrombolysis indicate a significant difference of at least P = .05. Bars represent group means with standard deviations. d-Dimer values for the four experimental groups appear inside the bars and directly reflect the results of thrombolysis.

Figure 5

Graph shows results of the first optimization experiment. TAP levels were increased from 20 to 80 W, and all remaining exposure parameters were kept constant at the levels used in the standard exposure experiments. Differing lowercase letters between mean levels of thrombolysis indicate a significant difference of at least P = .05. Data points represent group means with standard deviations.

Figure 6

Graph shows results of the second optimization experiment. The number of pulses per raster point was increased from 20 to 80, and all remaining exposure parameters were kept constant at the levels used in the standard exposure experiments. Differing lowercase letters between mean levels of thrombolysis indicate a significant difference of at least P = .05. Data points represent group means with standard deviations.

Figure 7

Simulation results achieved with a TAP of 20 W and a high-intensity focused ultrasound pulse of 100 msec in a clot show a nonuniform distribution of displacement in the lateral and axial dimensions. The peak displacement in the center of the focused ultrasound beam is just higher than 12 μm.