Efficacy of Sonothrombolysis Using Microbubbles Produced by a Catheter-Based Microfluidic Device in a Rat Model of Ischemic Stroke

  • Adam J. Dixon
  • Jun Li
  • John-Marschner Robert Rickel
  • Alexander L. Klibanov
  • Zhiyi Zuo
  • John A. HossackEmail author


Limitations of existing thrombolytic therapies for acute ischemic stroke have motivated the development of catheter-based approaches that utilize no or low doses of thrombolytic drugs combined with a mechanical action to either dissolve or extract the thrombus. Sonothrombolysis accelerates thrombus dissolution via the application of ultrasound combined with microbubble contrast agents and low doses of thrombolytics to mechanically disrupt the fibrin mesh. In this work, we studied the efficacy of catheter-directed sonothrombolysis in a rat model of ischemic stroke. Microbubbles of 10–20 µm diameter with a nitrogen gas core and a non-crosslinked albumin shell were produced by a flow-focusing microfluidic device in real time. The microbubbles were dispensed from a catheter located in the internal carotid artery for direct delivery to the thrombus-occluded middle cerebral artery, while ultrasound was administered through the skull and recombinant tissue plasminogen activator (rtPA) was infused via a tail vein catheter. The results of this study demonstrate that flow focusing microfluidic devices can be miniaturized to dimensions compatible with human catheterization and that large-diameter microbubbles comprised of high solubility gases can be safely administered intraarterially to deliver a sonothrombolytic therapy. Further, sonothrombolysis using intraarterial delivery of large microbubbles reduced cerebral infarct volumes by approximately 50% vs. no therapy, significantly improved functional neurological outcomes at 24 h, and permitted rtPA dose reduction of 3.3 (95% CI 1.8–3.8) fold when compared to therapy with intravenous rtPA alone.


Ischemic stroke Thrombolysis Ultrasound Microbubbles Microfluidics 



Partial support for this research is provided by the National Institutes of Health under NIH Grants S10 RR025594 and R01 HL141752 to JAH and by a NSF GRFP fellowship to AJD. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH or NSF.

Author Contributions

AJD, JAH, ZZ conceived of study. AJD wrote main manuscript text. AJD, JMRR, JL conducted experiments and analyzed data. JAH, ALK, and ZZ supervised the study and all authors reviewed the manuscript.

Conflict of Interest

Authors AJD and JAH are inventors listed on an issued patent (US Patent No. 9895,158) that relates to some aspects of the content of this study.18


  1. 1.
    Alexandrov, A. V., et al. Ultrasound-enhanced systemic thrombolysis for acute ischemic stroke. N Engl J Med 351:2170–2178, 2004.CrossRefGoogle Scholar
  2. 2.
    Alexandrov, A. V., et al. A pilot randomized clinical safety study of sonothrombolysis augmentation with ultrasound-activated perflutren-lipid microspheres for acute ischemic stroke. Stroke 39:1464–1469, 2008.CrossRefGoogle Scholar
  3. 3.
    Bekkers, S. C., S. K. Yazdani, R. Virmani, and J. Waltenberger. Microvascular obstruction: underlying pathophysiology and clinical diagnosis. J Am Coll Cardiol 55:1649–1660, 2010.CrossRefGoogle Scholar
  4. 4.
    Berkhemer, O. A., et al. A Randomized Trial of Intraarterial Treatment for Acute Ischemic Stroke. N Engl J Med 372:11–20, 2015.CrossRefGoogle Scholar
  5. 5.
    Bhatia, R., et al. Low rates of acute recanalization with intravenous recombinant tissue plasminogen activator in ischemic stroke: real-world experience and a call for action. Stroke 41:2254–2258, 2010.CrossRefGoogle Scholar
  6. 6.
    Borrelli, M. J., et al. Influences of microbubble diameter and ultrasonic parameters on in vitro sonothrombolysis efficacy. J Vasc Intervent Radiol 23:1677–1684, 2012.CrossRefGoogle Scholar
  7. 7.
    Broderick, J. P., et al. Endovascular therapy after intravenous t-PA versus t-PA alone for stroke. N Engl J Med 368:893–903, 2013.CrossRefGoogle Scholar
  8. 8.
    Brujan, E. A., T. Ikeda, and Y. Matsumoto. Jet formation and shock wave emission during collapse of ultrasound-induced cavitation bubbles and their role in the therapeutic applications of high-intensity focused ultrasound. Phys Med Biol 50:4797–4809, 2005.CrossRefGoogle Scholar
  9. 9.
    Brujan, E. A., and Y. Matsumoto. Collapse of micrometer-sized cavitation bubbles near a rigid boundary. Microfluid Nanofluid 13:957–966, 2012.CrossRefGoogle Scholar
  10. 10.
    Campbell, B. C. V., et al. Endovascular therapy for ischemic stroke with perfusion-imaging selection. N Engl J Med 372:1009–1018, 2015.CrossRefGoogle Scholar
  11. 11.
    Chen, J. L., A. H. Dhanaliwala, A. J. Dixon, A. L. Klibanov, and J. A. Hossack. Synthesis and characterization of transiently stable albumin-coated microbubbles via a flow-focusing microfluidic device. Ultrasound Med Biol 40:400–409, 2014.CrossRefGoogle Scholar
  12. 12.
    Choi, J. J., et al. Microbubble-size dependence of focused ultrasound-induced blood-brain barrier opening in mice in vivo. IEEE Trans Bio-med Eng 57:145–154, 2010.CrossRefGoogle Scholar
  13. 13.
    Crumrine, R. C., et al. Intra-arterial administration of recombinant tissue-type plasminogen activator (rt-PA) causes more intracranial bleeding than does intravenous rt-PA in a transient rat middle cerebral artery occlusion model. Exp Transl Stroke Med 3:10, 2011.CrossRefGoogle Scholar
  14. 14.
    Daffertshofer, M., et al. Efficacy of sonothrombolysis in a rat model of embolic ischemic stroke. Neurosci Lett 361:115–119, 2004.CrossRefGoogle Scholar
  15. 15.
    Dayton, P., J. Allen, and K. Ferrara. The magnitude of radiation force on ultrasound contrast agents. J Acoust Soc Am 112:2183–2192, 2002.CrossRefGoogle Scholar
  16. 16.
    Dhanaliwala, A. H., et al. In vivo imaging of microfluidic-produced microbubbles. Biomed Microdev 17:23, 2015.CrossRefGoogle Scholar
  17. 17.
    Dinia, M. R., M. Ribo, E. Santamarina, O. Maisterra, R. Delgado-Mederos, and J. Alvarez-Sabin, J. M., C. Molina,. Timing of microbubble-enhanced sonothrombolysis strongly predicts intracranial haemorrhage in acute ischaemic stroke. European Stroke Conference, 2008.Google Scholar
  18. 18.
    Dixon, A. J., and Hossack, J. A. Method and apparatus for accelerated disintegration of blood clot. United States of America Patent US9895158, 2018.Google Scholar
  19. 19.
    Dixon, A. J., A. H. Dhanaliwala, J. L. Chen, and J. A. Hossack. Enhanced intracellular delivery of a model drug using microbubbles produced by a microfluidic device. Ultrasound Med Biol 39:1267–1276, 2013.CrossRefGoogle Scholar
  20. 20.
    Dixon, A. J., J. M. R. Rickel, B. D. Shin, A. L. Klibanov, and J. A. Hossack. In vitro sonothrombolysis enhancement by transiently stable microbubbles produced by a flow-focusing microfluidic device. Ann Biomed Eng 46:222–232, 2018.CrossRefGoogle Scholar
  21. 21.
    Dixon, A. J., et al. Microbubble-mediated intravascular ultrasound imaging and drug delivery. IEEE Trans Ultrason Ferroelectr Freq Control 62:1674–1685, 2015.CrossRefGoogle Scholar
  22. 22.
    Fan, Z., H. Liu, M. Mayer, and C. X. Deng. Spatiotemporally controlled single cell sonoporation. Proc Natl Acad Sci USA 109:1–4, 2012.CrossRefGoogle Scholar
  23. 23.
    Gao, S., et al. Improvements in cerebral blood flow and recanalization rates with transcranial diagnostic ultrasound and intravenous microbubbles after acute cerebral emboli. Invest Radiol 49:593–600, 2014.CrossRefGoogle Scholar
  24. 24.
    Garstecki, P., et al. Formation of monodisperse bubbles in a microfluidic flow-focusing device. Appl Phys Lett 85:2649–2651, 2004.CrossRefGoogle Scholar
  25. 25.
    Goyal, M., et al. Randomized assessment of rapid endovascular treatment of ischemic stroke. N Engl J Med 372:1019–1030, 2015.CrossRefGoogle Scholar
  26. 26.
    Helfield, B., X. Chen, S. C. Watkins, and F. S. Villanueva. Biophysical insight into mechanisms of sonoporation. Proc Natl Acad Sci USA 113:9983–9988, 2016.CrossRefGoogle Scholar
  27. 27.
    Helps, S. C., M. Meyer-Witting, P. L. Reilly, and D. F. Gorman. Increasing doses of intracarotid air and cerebral blood flow in rabbits. Stroke 21:1340–1345, 1990.CrossRefGoogle Scholar
  28. 28.
    Hettiarachchi, K., E. Talu, M. L. Longo, P. A. Dayton, and A. P. Lee. On-chip generation of microbubbles as a practical technology for manufacturing contrast agents for ultrasonic imaging. Lab Chip 7:463, 2007.CrossRefGoogle Scholar
  29. 29.
    Jayaraman, M. V., J. A. Grossberg, K. M. Meisel, A. Shaikhouni, and B. Silver. The clinical and radiographic importance of distinguishing partial from near-complete reperfusion following intra-arterial stroke therapy. Am J Neuroradiol 34:135–139, 2013.CrossRefGoogle Scholar
  30. 30.
    Jovin, T. G., et al. Thrombectomy within 8 hours after symptom onset in ischemic stroke. N Engl J Med 372:2296–2306, 2015.CrossRefGoogle Scholar
  31. 31.
    Kilroy, J. P., et al. Reducing neointima formation in a swine model with IVUS and sirolimus microbubbles. Ann Biomed Eng 43:2642–2651, 2015.CrossRefGoogle Scholar
  32. 32.
    Kim, J., et al. Intravascular forward-looking ultrasound transducers for microbubble-mediated sonothrombolysis. Sci Rep 7:3454, 2017.CrossRefGoogle Scholar
  33. 33.
    Li, L., and Z. Zuo. Isoflurane preconditioning improves short-term and long-term neurological outcome after focal brain ischemia in adult rats. Neuroscience 164:497–506, 2009.CrossRefGoogle Scholar
  34. 34.
    Longuet-Higgins, M. S. Viscous streaming from an oscillating spherical bubble. Proc R Soc Lond A 454:725–742, 1998.CrossRefGoogle Scholar
  35. 35.
    Marler, J. Tissue plasminogen activator for acute ischemic stroke. N Engl J Med 333:1581–1588, 1995.CrossRefGoogle Scholar
  36. 36.
    Marmottant, P., and S. Hilgenfeldt. Controlled vesicle deformation and lysis by single oscillating bubbles. Nature 423:153–156, 2003.CrossRefGoogle Scholar
  37. 37.
    Marmottant, P., M. Versluis, N. de Jong, S. Hilgenfeldt, and D. Lohse. High-speed imaging of an ultrasound-driven bubble in contact with a wall: “Narcissus” effect and resolved acoustic streaming. Exp Fluids 41:147–153, 2006.CrossRefGoogle Scholar
  38. 38.
    Mathias, Jr, W., et al. Diagnostic ultrasound impulses improve microvascular flow in patients with STEMI receiving intravenous microbubbles. J Am Coll Cardiol 67:2506–2515, 2016.CrossRefGoogle Scholar
  39. 39.
    Molina, C. A., et al. Microbubble administration accelerates clot lysis during continuous 2-MHz ultrasound monitoring in stroke patients treated with intravenous tissue plasminogen activator. Stroke 37:425–429, 2006.CrossRefGoogle Scholar
  40. 40.
    Molina, C. A., et al. Transcranial ultrasound in clinical sonothrombolysis (TUCSON) trial. Ann Neurol 66:28–38, 2009.CrossRefGoogle Scholar
  41. 41.
    Porter, T. R., et al. The thrombolytic effect of diagnostic ultrasound-induced microbubble cavitation in acute carotid thromboembolism. Invest Radiol 52:477–481, 2017.CrossRefGoogle Scholar
  42. 42.
    Postema, M., P. Marmottant, C. T. Lancee, S. Hilgenfeldt, and N. de Jong. Ultrasound-induced microbubble coalescence. Ultrasound Med Biol 30:1337–1344, 2004.CrossRefGoogle Scholar
  43. 43.
    Powers, W. J., et al. 2015 AHA/ASA focused update of the 2013 guidelines for the early management of patients with acute ischemic stroke regarding endovascular treatment: a guideline for healthcare professionals from the American Heart Association/American Stroke Association. Stroke 46:3020–3035, 2015.CrossRefGoogle Scholar
  44. 44.
    Rickel, J. M. R., A. J. Dixon, A. L. Klibanov, and J. A. Hossack. A flow focusing microfluidic device with an integrated Coulter particle counter for production, counting and size characterization of monodisperse microbubbles. Lab Chip 18:2653–2664, 2018.CrossRefGoogle Scholar
  45. 45.
    Sakuma, T., H. Leong-Poi, N. G. Fisher, N. C. Goodman, and S. Kaul. Further insights into the no-reflow phenomenon after primary angioplasty in acute myocardial infarction: the role of microthromboemboli. J Am Soc Echocardiogr 16:15–21, 2003.CrossRefGoogle Scholar
  46. 46.
    Saver, J. L., et al. Stent-retriever thrombectomy after Intravenous t-PA vs. t-PA alone in stroke. N Engl J Med 372:2285–2295, 2015.CrossRefGoogle Scholar
  47. 47.
    Schleicher, N., et al. Sonothrombolysis with BR38 microbubbles improves microvascular patency in a rat model of stroke. PLoS ONE 11:e0152898, 2016.CrossRefGoogle Scholar
  48. 48.
    Schwamm, L. H., et al. Temporal trends in patient characteristics and treatment with intravenous thrombolysis among acute ischemic stroke patients at get with the guidelines-stroke hospitals. Circ Cardiovasc Qual Outcomes 6:543–549, 2013.CrossRefGoogle Scholar
  49. 49.
    Shortencarier, M. J., et al. A method for radiation-force localized drug delivery using gas-filled lipospheres. IEEE Trans Ultrason Ferroelectr Freq Control 51:822–831, 2004.CrossRefGoogle Scholar
  50. 50.
    Talu, E., et al. Maintaining monodispersity in a microbubble population formed by flow-focusing. Langmuir 24:1745–1749, 2008.CrossRefGoogle Scholar
  51. 51.
    Tissue plasminogen activator for acute ischemic stroke. The National Institute of Neurological Disorders and Stroke rt-PA Stroke Study Group. N Engl J Med 333:1581–1587, 1995.CrossRefGoogle Scholar
  52. 52.
    Tomsick, T. TIMI, TIBI, TICI: I Came, I saw, I got confused. AJNR Am J Neuroradiol 28:382–384, 2007.CrossRefGoogle Scholar
  53. 53.
    van Wamel, A., et al. Vibrating microbubbles poking individual cells: drug transfer into cells via sonoporation. J Control Release 112:149–155, 2006.CrossRefGoogle Scholar
  54. 54.
    Wu, J. Theoretical study on shear stress generated by microstreaming surrounding contrast agents attached to living cells. Ultrasound Med Biol 28:125–129, 2002.CrossRefGoogle Scholar
  55. 55.
    Wu, J. H., and S. L. Diamond. Tissue plasminogen activator (tPA) inhibits plasmin degradation of fibrin. A mechanism that slows tPA-mediated fibrinolysis but does not require alpha 2-antiplasmin or leakage of intrinsic plasminogen. J Clin Invest 95:2483–2490, 1995.CrossRefGoogle Scholar
  56. 56.
    Yueh-Hsun, C., C. Po-Wen, and L. Pai-Chi. Combining radiation force with cavitation for enhanced sonothrombolysis. IEEE Trans Ultrason Ferroelectr Freq Control 60:97–104, 2013.CrossRefGoogle Scholar

Copyright information

© Biomedical Engineering Society 2019

Authors and Affiliations

  1. 1.Department of Biomedical EngineeringUniversity of VirginiaCharlottesvilleUSA
  2. 2.Department of AnesthesiologyUniversity of Virginia School of MedicineCharlottesvilleUSA
  3. 3.Cardiovascular MedicineUniversity of Virginia School of MedicineCharlottesvilleUSA

Personalised recommendations