[Effect of diagnostic ultrasound-mediated microbubble contrast and urokinase on augmentation thrombolysis and optimization of the major parameters: an in vitro study].

To investigate the efficacy of diagnostic ultrasound and microbubble contrast (MB) on enhancing thrombolysis in combination with urokinase (UK) and to determine the optimal combination for thrombolysis in vitro.Four types of standardized red thrombus were prepared in vitro, including 3-hour-old (3 h), 6-hour-old (6 h), 12-hour-old (12 h), and 24-hour-old (24 h). The major parameters for the designed experiments included transmit powers of ultrasound (factor A, 5%, 25%, 50%, 100%), MB volumes (factor B, 50 microL, 100 microL, 200 microL, 400 microL), UK concentrations (factor C, 100 U/mL, 200 U/mL, 400 U/mL, 800 U/mL), and lysis time (factor D, 10 min, 20 min, 30 min, 40 min). An orthogonal array experimental design (OAD) based on four levels L16 (4(5)) of the above four parameters was employed to optimize the thrombolysis conditions. During the procedure of thrombolysis, the diagnostic ultrasound frequency was fixed at 1.82 MHz. The histopathological changes measured by HE staining and scanning electron microscope (SEM) were carried out to observe the clots before and after thrombolysis. The loss of clot weight before and after treatment was measured to determine the lysis efficiency (LE). Analysis of variance (ANOVA) was performed to assess the LE according to the L16 (4(5)) matrix.The HE staining and SEM observation of thrombolysis under the following experimental conditions of 5% ultrasound transmit power, 400 microL MB volume, 800 U/mL UK concentration, and 40 min lysis time showed remarkable disaggregation of fibrin nets. The above four factors had significant impact on thrombus (all P < 0.05), among which UK concentrations (factor C) was the most significant one. The optimal scheme was determined as a C4-D4-A1-B4 mode, with UK concentration 800 U/mL, lysis time 40 min, transmit power 5%, and MB volume 400 microL, respectively. The LE curves for 3h clots were superior to the others. The lysis efficiencies for the clots showed significant differences among different type of thrombus (all P < 0.05).1.82 MHz diagnostic ultrasound and microbubble contrast can be applied to augment thrombolysis in vitro even with a transmit power as low as 5%. Under the condition of fixed ultrasound frequency, the LE of thrombus increase with increased UK concentrations, lysis time and MB volumes, and decrease with increased thrombus ages.