As revealed by the analysis of clots retrieved from ischemic

As revealed by the analysis of clots retrieved from ischemic stroke patients (Liebeskind et al. 2011; Marder et al. 2006), the main components of clots are red blood acetanilide (RBC), fibrin and platelets. An important issue in the clot lysis process is the effect of STL on the fibrin network. Fibrin strands provide a 3-D scaffold for the intravascular clot and ensure its stability against mechanical stress and degradation (Mosesson 2005; Weisel 2007). Therefore, degradation of the fibrin network is a key factor in the success of STL therapy, and its monitoring is essential.
In a previous study (Petit et al. 2015), we used two different quantification techniques to assess fibrin degradation: measurement of radiolabeled-fibrin degradation products (FDP) and D-dimer assay. It was found that at high acoustic pressure, the combination of ultrasound (US) and MB was not able to degrade the fibrin network in the absence of a thrombolytic drug. On the contrary, a clear synergistic effect was observed on fibrin degradation when US, MB and recombinant tissue plasminogen activator (rtPA) were associated.
Even though the respective roles of microstreaming and microjets in MB-enhanced STL remain unclear, it is commonly recognized that cavitation-based phenomena play an important role in the clot dissolution process, as reported by some authors (Datta et al. 2008; Hitchcock et al. 2011; Prokop et al. 2007; Shi et al. 2010; Xie et al. 2011). Indeed the argument for using MB to enhance US action is that MB dramatically reduce the acoustic cavitation threshold by providing cavitation nuclei in the medium. Cavitation could induce direct mechanical damage to the clot as well as positively affect the action of rtPA by improving access to fibrin strands and drug transport. For example, Prokop et al. (2007) reported that in the presence of a thrombolytic drug, the enhancement of clot lysis is related to the cavitation activity caused by the combination of US and MB. Historically, cavitation activity has been classified as either stable or inertial (Neppiras 1980). Stable cavitation (SC) designates the stable oscillation of MB over time in response to applied acoustic waves (Miller et al. 1996) and induces microstreaming (Collis et al. 2010; Leighton 1994; Wu and Nyborg 2008). At higher acoustic pressures, inertial cavitation (IC) occurs, and MB undergo a rapid increase in size followed by violent bubble collapse (Miller et al. 1996). Inertial cavitation produces a more violent mechanical action, such as microjets, but within a much shorter period than SC action (Holland and Apfel 1990; Miller et al. 1996).
Whether SC or IC would be more appropriate for STL is still debated. For instance, Datta et al. (2008) observed a significant correlation between clot lysis and SC activity with 120-kHz US and MB. Hitchcock et al. (2011) later reported significant enhancement of rtPA-induced lysis by US and MB, with US parameters selected to have maximal SC exposure. Thus, these studies emphasize the contribution of SC in MB-enhanced STL. Although the experiments were designed to maximize SC phenomena, IC was also present in these studies, which underlines the complexity of discriminating the effect of each type of cavitation.


For all experiments, clot lysis was assessed as diameter loss (mm) and 125I-FDP release (%) after 60 min of treatment. The results are illustrated in Figure 2(a, b).
For the control (plasma only), no significant diameter loss (0.02 ± 0.01 mm) or 125I-FDP release (0.3 ± 0.1%) was observed. This was a sign of good clot stability for the duration of the experiment. In the group exposed to rtPA alone, a diameter loss of 0.39 ± 0.01 mm was measured, with a corresponding 125I-FDP release of 51.7 ± 2.0%, this being the mark of efficient enzymatic fibrinolysis.
Figure 2a also illustrates that for all conditions combining rtPA + US + MB, diameter loss was significantly increased compared with that for rtPA alone. Examples of typical treated clots are illustrated in Figure 3a. Considering fibrin degradation (Fig. 2b), under the condition of SC only (200 kPa, 500 ms ON/750 ms OFF), no enhancement of 125I-FDP release was observed (53.2 ± 0.9%, p > 0.05 vs. rtPA). Under the condition of coexisting SC and IC (350 kPa, 100 ms ON/1,150 ms OFF), fibrin degradation was significantly enhanced compared with rtPA (57.2 ± 2.9%, p < 0.001). Additionally, histological analysis revealed multiple small areas lacking RBC in the area exposed to US (Fig. 4c). Interestingly, at 350 kPa, when the pulse length was reduced from 100 to 1 ms, the increase in fibrin degradation provided by US + MB was lost (50.9 ± 1.9%, p > 0.05 vs. rtPA). Finally, at 1,300 kPa (1 ms ON/1,249 ms OFF), inducing essentially IC, there was an absolute increase of 15% (p < 0.001 versus rtPA) in fibrin degradation. Note that the lysis was almost complete in the central part of the clot (Fig. 3a).