Mediation of long-pulsed ultrasound enhanced microbubble recombinant tissue plasminogen activator thrombolysis in a rat model of platelet-rich thrombus

Introduction

In about 5–20% of patient who experience acute ST-segment elevation myocardial infarction (STEMI), successful revascularization of the epicardial coronary artery via percutaneous coronary intervention (PCI) cannot provide ideal microvascular reperfusion (1-3). Insufficient postprocedural reperfusion in myocardial capillaries is mainly caused by microvascular embolization and remains an important limitation that substantially reduces the beneficial effects of PCI. Microvascular embolization in patients is usually associated with a higher incidence of malignant arrhythmia, refractory heart failure, progressive left ventricular remodeling, and sudden death. Ultrasound (US)-enhanced microbubble (MB) therapy has been investigated as a useful technique for the lysis of occlusive thrombi in acute myocardial infarction (4,5) and stroke (6). The therapeutic effects of US-assisted MB therapy have been found to facilitate thrombolysis under different experimental conditions: with recombinant tissue plasminogen activator (rt-PA) (7-9) and without rt-PA (10-12), with low frequency (40–500 kHz) (8,13) or high frequency (1–5 MHz) (14,15), and with targeted MB (5) or untargeted MB (16-18). Presently, the US used in clinic is usually short cycle (≤5 µs) (19). Long-pulsed duration (20 µs) US with MBs has become a valuable adjunct to reperfusion therapy for improving the effectiveness of sonothrombolysis therapy at a lower mechanical index (20). One clinical trial demonstrated that guided high mechanical index impulses with a 20-µs long-pulsed duration were able to prevent microvascular obstruction when coadministered with MB infusion (4). In other research, 30-µs pulse-length US was used to augment limb perfusion in patients with peripheral artery disease (21,22). Different from short-pulsed US, different long-pulse lengths (100–5,000 µs) of US were found to dissolve the microthrombi successfully in vitro (17). Further experiments showed long-tone-burst (5,000 µs)-assisted MB therapy to be an important strategy for the treatment of thrombotic microvascular obstruction in vivo (11). In a rodent model of microvascular obstruction, longer pulses (5,000 µs) caused greater MB cavitation followed by a more rapid microvascular flow restoration than did a 10-µs pulse US (23). Ultrahigh-speed imaging suggests that acoustic radiation forces (Bjerknes forces) can help mediate clot disruption under inertial cavitation conditions (24). The better treatment effects of a long-pulse duration of therapeutic impulses may be attributed to the sustained cavitation events that could restore both microvascular and epicardial flow, suggesting that long-pulse US is a promising direction for improving sonothrombolysis. rt-PA has been used for the treatment of STEMI as reperfusion therapy, but it does not provide an ideal therapeutic effect for arterial patency and is associated with disadvantages of cerebral or gastrointestinal bleeding (25). Transcatheter thrombolysis therapy decreases the risk of bleeding, but it’s expensive and limited in large medical centers. Holland et al. demonstrated that both 120-kHz and 1-MHz pulsed or and continuous-wave US can enhance rt-PA thrombolysis (26) and that the initial lytic rate increases linearly with the duty cycle. US facilitates the transport of fibrinolytic enzymes into the clot by increasing the transport of reactants through a cavitation-related mechanism, and stable cavitation causing MB oscillation and microstreaming is critical to this process (8). This combined therapy has also been demonstrated to treat microvascular embolization and reperfuse the capillary network for infarction with no reflow (27).

US-assisted MB-mediated rt-PA thrombolysis therapy is still not widely used by the limitation of MB cavitation effect, because arterial total occlusion restricts blood flow and causes only a portion of the MBs arriving at the surface of the thrombi. In this study, a model of platelet-rich thrombus with total carotid arterial occlusion was established, and the synergistic effects of a novel 1,000 µs-pulse-length US, MB, and rt-PA were evaluated. We present this article in accordance with the ARRIVE reporting checklist (available at https://cdt.amegroups.com/article/view/10.21037/cdt-23-356/rc).

Methods

Preparation of rt-PA and MB

rt-PA was stored at −80 ℃ in a freezer and was dilated with saline to a concentration of 1 mg/mL (28) before intravenous infusion. A dosage of 10 mg/kg was selected based on previous rat species studies (7,26). rt-PA was administered with a 10% bolus, which was followed by a 90% infusion with a syringe pump at a constant rate. Lipid MB (Sonovue, Bracco, Italy) was mixed with saline and then injected at a dose of 0.1 mL every 3 min. A Coulter counter showed the mean diameter of MB was 2.46±0.05 µm, with a concentration of (2.99±0.19)×10⁹/mL. To ensure the MBs arrived at the thrombi, visualization was performed with a 18L7 imaging probe from an Aplio 500 US machine (Toshiba, Tokyo, Japan).

Animal model

Experiments were performed under a project license granted by ethics review board of Jinan University, Guangzhou, China (No. 44002100033991), in compliance with the institutional guidelines for the care and use of animals. To begin, 42 Sprague-Dawley (SD) male rats (n=6 for each group) weighing 290±30 g (age of 8–9 weeks) from the university animal center were anesthetized with pentobarbital sodium (40 mg/kg body weight, i.p). By using a computer based random order generator, all the rats were randomly numbered, and the groups were determined by a random draw. Rats were placed on a thermostat plate kept at a constant temperature of 37 ℃. The right external jugular vein was cannulated with a Polyethylene 50 tubing for infusion of rt-PA and MBs. The rat total carotid arterial occlusion model was established with 10% ferric chloride (FeCl₃) to induce platelet-rich thrombi (29). A segment of the left carotid artery was exposed using blunt dissection, and a plastic sheeting was placed beneath the carotid artery for protection of the surrounding tissues. A piece of paper (1 cm × 1 cm) saturated with 10% FeCl₃ solution was placed between the carotid artery and plastic sheeting to form thrombi (Figure S1). After 10 min of exposure, the filter paper and plastic sheeting were both removed.

The peak systolic velocity (PSV) was measured just proximal to the site of the injury in the left carotid artery with the Aplio 500 US machine. For reflection of the distal arterial resistance, the resistance index (RI) was derived with the following equation: RI = (PSV – diastolic flow velocity)/PSV. The two-dimensional (2D) and pulse-wave Doppler images of the left carotid artery at three different stages (baseline, thrombus, thrombolysis) were collected. No blood flow in Doppler US implied successful total occlusion of the carotid artery. Carotid arterial recanalization grade was assessed according to the following scheme: no flow across the lesion, grade 0; arterial stenosis >50%, grade I; residual arterial stenosis <50%; and complete recanalization, grade II. Individuals who died from anesthetic accident were excluded from our study.

Therapeutic US

A 1-MHz single-element transducer (A302S, Olympus, Tokyo, Japan) driven by a function generator (33250A, Agilent Technologies, Inc., Santa Clara, CA, USA) and a power amplifier (2200L, Electronics & Innovation, Rochester, NY, USA) was used to generate the therapeutic US (Figure S2). Because carotid arteries of the rats are superficial and because there is less attenuation for the application of US, 0.6 MPa (peak negative pressure) was used. Most of the recent related studies used 10- to 30-µs pulse lengths (4,20,21); however, the use of 5,000 µs has also drawn considerable attention (11,23), and therefore a 1,000-µs pulse length was selected for this investigation. Subsequently, 1-MHz US was delivered with two different duty cycles: 0.03% (pulse interval 3 s) and 10% (pulse interval 10 ms). When platelet-rich thrombus was successfully induced by FeCl₃, the transducer was gently placed vertically above the left carotid artery. We made sure that the thrombi were entirely covered by therapeutic US before we gently moved the probe up and down.

Treatment protocol

A protocol was prepared before the study without registration. SD rats were randomly divided into seven groups (n=6 for each group): (I) control; (II) rt-PA; (III) high duty cycle US + MB; (IV) low duty cycle US + rt-PA; (V) high duty cycle US + rt-PA; (VI) low duty cycle US + rt-PA + MB; and (VII) high duty cycle US + rt-PA + MB. n=6 for each group, and the estimation of sample size was based on a previous unpublished study. For all groups, the 2D and pulse-wave Doppler images of three different stages (baseline, thrombus, thrombolysis) were collected (Figure S3). The PSV, RI and arterial recanalization grade were calculated (Tables S1,S2). During the allocation, the conduct of the experiment, the outcome assessment, and the data analysis, were completed without the knowledge of each other.

Histology

Finally, rats were sacrificed via an overdose of potassium chloride, and hematoxylin and eosin (HE) staining was performed to analyze the effects of long-tone-burst US. The carotid arterial segment exposed to therapeutic US was selected, and the territory exposed to therapeutic US was sampled for histology. The magnification factor was 100×, and the representative pictures were selected.

Statistical analysis

SPSS 26.0 (IBM Corp., Armonk, NY, USA) was used for statistical analysis, and the PSV values are expressed as the mean ± standard deviation. One-way analysis of variance was used to compare differences within groups, the Bonferroni post hoc test was used for the comparison of the differences between groups, and the Fisher exact test was used to analyze the recanalization rate. A two-sided P value <0.05 was considered significant.

Results

A successful rat model of total carotid arterial occlusion was induced with a 10% FeCl₃ solution. As shown in Figure 1A, color Doppler confirmed the carotid artery was totally occluded by the fresh thrombus. Pathology showed the thrombi were full of insoluble fibrous tissue, forming dense clots, with only a small number of red blood cells. Figure 1B shows how the pure administration of rt-PA did not have thrombolytic effect for the fresh platelet-rich thrombi. However, when assisted with low duty cycle US, rt-PA showed good thrombolysis (P=0.01 vs. control). Moreover, there was a tendency toward an increase in thrombolytic rate when MBs were further added. Under the high duty cycle US condition, pure MBs did not show enough capacity to dissolving platelet-rich thrombi. However, US combined with rt-PA showed apparent thrombolysis (P=0.01 vs. control). Furthermore, there was an increase in the thrombolytic rate in the US + rt-PA + MB group (P<0.001 vs. control).

Figure 1 Platelet-rich thrombus and the thrombolytic effects. (A) Platelet-rich thrombi induced by 10% FeCl₃ solution in a rat model. (Left) the carotid artery was totally occluded by the fresh thrombi (red arrow). (Right) hematoxylin and eosin staining showed that the fresh platelet-rich thrombi were full of insoluble fibrous tissue, forming dense clots (black arrow) with only a small number of red blood cells (red arrow). (B) Recanalization after thrombolytic therapies. (Left) rt-PA showed no thrombolytic effects, but low duty cycle US + rt-PA showed good thrombolysis. (Right) high duty cycle US + rt-PA + MB showed the best thrombolytic effects. n=6 for each group. CON, control; rt-PA, recombinant tissue plasminogen activator; US(L), low duty cycle ultrasound; MB, microbubble; US(H), high duty cycle ultrasound; US, ultrasound.

HE staining of formalin-preserved arterial slices confirmed the successful establishment of the platelet- rich thrombi. Most of the thrombi remained intact in the control, rt-PA, and US + MB groups, as illustrated in Figure 2. A fairly large percent of thrombi was dissolved in the low duty cycle US + rt-PA and high duty cycle US + rt-PA groups. The thrombolytic effects were further intensified in the low duty cycle US + rt-PA + MB and high duty cycle US + rt-PA + MB groups.

Figure 2 Hematoxylin and eosin staining of the platelet-rich thrombi in rats post thrombolytic therapy (magnification factor 100×). There was abundant insoluble fibrous tissue in the vessels, forming dense clots in control, rt-PA, and high duty-cycle ultrasound + microbubble groups. Only a small amount of insoluble latticed fibrous tissues was found in the ultrasound + rt-PA + microbubble groups. The US(L) + rt-PA group was in the intermediate stage. The blue arrow indicates unsuccessful thrombolysis, and the red arrow indicates successful thrombolysis. CON, control; rt-PA, recombinant tissue plasminogen activator; US(H), high duty cycle ultrasound; MB, microbubble; US(L), low duty cycle ultrasound.

Discussion

In this study, platelet-rich thrombi in a rat model of total carotid arterial occlusion was made, and the synergistic effects of long-pulsed US, rt-PA, and MB were evaluated. FeCl₃ with different concentrations (2–60%) has been widely used to induce arterial thrombosis in a variety of species (30). FeCl₃ causes endothelial injury, platelet aggregation, thrombin release, and fibrin deposition, along with dense-structure thrombus. The intrinsic components of FeCl₃-induced thrombus consist of activated platelets, fibrin, and red blood cells, which is a similar composition to that of human coronary arteries after acute myocardial infarction. In our experiment, 10% FeCl₃ was used with an exposure time of 10 min. Color Doppler confirmed total occlusion in left carotid arteries in all the rats, and the success rate of the arterial thrombosis model was 100%. Therefore, our procedure is a simple, sensitive, and reproducible method of establishing platelet-rich thrombi in a rat model.

To our knowledge, this is the first study to show long-pulsed US-mediated successful thrombolysis for platelet-rich thrombus in a totally occluded arterial model. Most of the preliminary studies in vitro involve red blood cell-rich thrombi, with MBs being continuously infused and arriving at the location of the thrombus formation (8,9). Therefore, a persistent MB cavitation effect can lead to thrombolysis while therapeutic US is delivered without attenuation. However, new-formed thrombi usually cause arterial total occlusion and restrict blood flow in patients with STEMI, lead to only a portion of the MBs arriving at the surface of the thrombi. A decreased MB cavitation effect often result in the failures of sonothrombolysis, especially for the platelet-rich thrombi with a dense structure. In our study, ~0.5 mL of MBs were used, and the synergistic effects of MBs and rt-PA showed excellent thrombolytic capacity under long-pulsed acoustic conditions.

rt-PA is an important treatment drug and has been approved by the US Food and Drug Administration (FDA) for ischemic stroke (31). The stirring effect caused by acoustic microstreaming is of the main mechanisms underlying the action of US-enhanced MB-mediated rt-PA thrombolysis since it facilitates the exposure of new binding sites for plasmin near the fibrin mesh, accelerates oscillating MBs, and promotes removal of fibrin degradation products away from the clot (8). Stable cavitation has been demonstrated to accelerate thrombolysis when US, rt-PA, and MB act synergistically (8) (Figure S4). In our study, pure administration of rt-PA did not cause patency of the occluded artery likely because the platelet-rich thrombi were quite solid. High duty cycle US (0.6 MPa) with MBs typically generates stable cavitation events; (17) therefore, the radiation forces from microstreaming are unable to completely dissolve the rigid thrombi. Additionally, MBs, acting synergistically with rt-PA, can improve the recanalization rate. In our study, the recanalization grade in the US + rt-PA + MB group was higher than that in the US + rt-PA group regardless of which duty cycle was used. The duty cycle dependence of 120-kHz US-enhanced thrombolysis was demonstrated in a human clot model (13). With this model, the lytic rate increased linearly with the increase of the duty cycle since the increase of duty cycle provides more sustained MB cavitation events that can accelerate thrombolysis. Our study found that in rats treated with a treatment of US + rt-PA + MB, the high duty cycle achieved a 100% recanalization rate after thrombolytic therapy. A recent study examined MBs undergoing inertial cavitation under 5,000-µs pulse-length US with high acoustic pressure (32). Under the long-tone-burst US excitation, MB oscillations activated the endothelial nitric oxide synthase (eNOS) pathway with nitro oxide playing a significant role, and blood reperfusion was improved (33). In a different study, the MB acoustic behaviors caused sustained enhancement of cellular and vascular permeability through a physical, cavitation-based mechanism of sonoporation (34). In our study, long-pulse-length US of 1,000 µs was used with MB-assisted rt-PA therapy and showed highly promising effects in dissolving clots. The passive detection of MB cavitation should be monitored for further clarification of the thrombolytic mechanisms with long-tone-burst US.

Strengths and limitations

First of all, the thrombus in our model was full of fresh platelets with dense structure, resistant to pure intravenous administration of rt-PA. Moreover, a novel 1,000 µs-pulse-length was used, and this long-pulsed US showed powerful thrombolytic effects in a rat model of platelet-rich thrombus. Third, the successful thrombolysis in the total carotid arterial occlusion model showed this combined therapy has great potentials to overcome the shortcoming of traditional US-assisted MB-mediated rt-PA therapy—limitation of cavitation effect. The main limitation of our study is the detection of cavitation activities (Figure S5) was not recorded. Further studies are needed to clarify of the intrinsic mechanisms of this promising combined therapy.

Conclusions

Long-tone-burst US-assisted MB-enhanced rt-PA therapy showed successful thrombolytic effects for platelet-rich thrombus in rats with total arterial occlusion and may offer a powerful approach for the treatment for arterial thrombosis.

Acknowledgments

Funding: This study was funded by the National Natural Science Foundation of China (grant No. 81400266 to X. Chen) and the counterpart fund from the First Affiliated Hospital of Jinan University, Guangzhou, China (grant No. 501134 to X. Chen). The funders have no role in the design and conduct of the study; collection, analysis, and interpretation of the data; preparation, or approval of the manuscript.

Footnote

Reporting Checklist: The authors have completed the ARRIVE reporting checklist. Available at https://cdt.amegroups.com/article/view/10.21037/cdt-23-356/rc

Data Sharing Statement: Available at https://cdt.amegroups.com/article/view/10.21037/cdt-23-356/dss

Peer Review File: Available at https://cdt.amegroups.com/article/view/10.21037/cdt-23-356/prf

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (Available at https://cdt.amegroups.com/article/view/10.21037/cdt-23-356/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. Experiments were performed under a project license granted by ethics review board ofJinan University, Guangzhou, China (No. 44002100033991), in compliance with the institutional guidelines for the care and use of animals.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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