An Echogenic Clot Method for Thrombolysis Monitoring in Thrombotic Stroke Models
Main Article Content
Abstract
To demonstrate thrombolytic efficacy of a tissue plasminogen activator (tPA)-loaded echogenic liposome (TELIP) formulation in a rabbit thrombotic stroke model (the most relevant animal model for evaluation of directed thrombolytic therapy for ischemic stroke), we sought to develop a means of monitoring thrombus dissolution quantitatively by ultrasound imaging methods. We hypothesized that a gas-free ultrasound contrast agent can be incorporated into blood clots at a concentration that does not affect the tPA-mediated clot dissolution rate, while enabling quantitative assessment of the clot dissolution rate. Clots were formed from a mixture of whole rabbit blood, 1 M calcium chloride, human thrombin and varying amounts of microcrystalline cellulose. Washed clots in tubes were weighed at 30, 60 and 90 minutes after addition of recombinant tPA (rtPA) in porcine plasma (100 µg/ml). Clot echogenicity at each time point was assessed using a Philips HDI 5000 ultrasound system using an L12-5 linear array probe. Recorded Images underwent videodensitometric analysis that converted image reflectivity to mean gray scale values (MGSV). We found that 1.12 mg/ml of microcrystalline cellulose in rabbit blood clots (0.2 ml) provided optimal echogenicity without affecting clot dissolution rates (0.3-0.6 mg/min.) caused by rtPA. The clot dissolution rate measured by videodensitometric analysis of the echogenic clots agreed well with that determined by mass loss measurements (0.28% 0-time value/minute). This method will be important for demonstrating in vivo efficacy with potentially decreased hemorrhagic effects provided by directed tPA vehicles relative to systemic administration of the free thrombolytic.
Article Details
How to Cite
CARTER, Dalton E. et al.
An Echogenic Clot Method for Thrombolysis Monitoring in Thrombotic Stroke Models.
Medical Research Archives, [S.l.], v. 11, n. 3, mar. 2023.
ISSN 2375-1924.
Available at: <https://esmed.org/MRA/mra/article/view/3702>. Date accessed: 25 apr. 2024.
doi: https://doi.org/10.18103/mra.v11i3.3702.
Section
Research Articles
The Medical Research Archives grants authors the right to publish and reproduce the unrevised contribution in whole or in part at any time and in any form for any scholarly non-commercial purpose with the condition that all publications of the contribution include a full citation to the journal as published by the Medical Research Archives.
References
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8. Sakharov DV, Rijken DC. Superficial accumulation of plasminogen during plasma clot lysis. Circulation. Oct 1 1995;92(7):1883-90.
9. Daffertshofer M, Gass A, Ringleb P, et al. Transcranial low-frequency ultrasound-mediated thrombolysis in brain ischemia: increased risk of hemorrhage with combined ultrasound and tissue plasminogen activator: results of a phase II clinical trial. Clinical Trial, Phase II Multicenter Study
Research Support, Non-U.S. Gov't. Stroke; a journal of cerebral circulation. Jul 2005;36(7):1441-6. doi:10.1161/01.STR.0000170707.86793.1a
10. Graham GD. Tissue plasminogen activator for acute ischemic stroke in clinical practice: a meta-analysis of safety data. Meta-Analysis Research Support, U.S. Gov't, P.H.S. Stroke; a journal of cerebral circulation. Dec 2003;34(12):2847-50. doi:10.1161/01.STR.0000101752.23813.C3
11. Katzan IL, Hammer MD, Hixson ED, Furlan AJ, Abou-Chebl A, Nadzam DM. Utilization of intravenous tissue plasminogen activator for acute ischemic stroke. Comparative Study. Archives of neurology. Mar 2004;61(3):346-50. doi:10.1001/archneur.61.3.346
12. Liang BA, Lew R, Zivin JA. Review of tissue plasminogen activator, ischemic stroke, and potential legal issues. Review. Archives of neurology. Nov 2008;65(11):1429-33. doi:10.1001/archneur.65.11.1429
13. Zaidat OO, Wolfe T, Hussain SI, et al. Interventional acute ischemic stroke therapy with intracranial self-expanding stent. Letter. Stroke; a journal of cerebral circulation. Aug 2008;39(8):2392-5. doi:10.1161/STROKEAHA.107.510966
14. Alexandrov AV, Molina CA, Grotta JC, et al. Ultrasound-enhanced systemic thrombolysis for acute ischemic stroke. Clinical Trial Clinical Trial, Phase II Multicenter Study Randomized Controlled Trial
Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S. The New England journal of medicine. Nov 18 2004;351(21):2170-8. doi:10.1056/NEJMoa041175
15. Huang S, Hamilton AJ, Tiukinhoy SD, et al. Liposomes as ultrasound imaging contrast agents and as ultrasound-sensitive drug delivery agents. Lectures. Cellular & molecular biology letters. 2002;7(2):233-5.
16. Huang SL. Liposomes in ultrasonic drug and gene delivery. Research Support, Non-U.S. Gov't
Review. Advanced drug delivery reviews. Jun 30 2008;60(10):1167-76. doi:10.1016/j.addr.2008.03.003
17. Tiukinhoy-Laing SD, Huang S, Klegerman M, Holland CK, McPherson DD. Ultrasound-facilitated thrombolysis using tissue-plasminogen activator-loaded echogenic liposomes. Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't. Thrombosis research. 2007;119(6):777-84. doi:10.1016/j.thromres.2006.06.009
18. Laing ST, Moody MR, Smulevitz B, et al. Doppler ultrasound enhances the thrombolytic activity of tissue plasminogen activator-loaded echogenic liposomes in vivo. Circulation. 2008;118(18):S643.
19. Moody M, Laing ST, Huang S, et al. Doppler ultrasound enhances the thrombolytic activity of tissue-plasminogen activator-loaded echogenic liposomes. Abstract. Circulation. October 16, 2007 2007;116(16):II-646.
20. Laing ST, Moody M, Smulevitz B, et al. Ultrasound-enhanced thrombolytic effect of tissue plasminogen activator-loaded echogenic liposomes in an in vivo rabbit aorta thrombus model--brief report. Research Support, N.I.H., Extramural. Arteriosclerosis, thrombosis, and vascular biology. Jun 2011;31(6):1357-9. doi:10.1161/ATVBAHA.111.225938
21. Laing ST, Moody MR, Kim H, et al. Thrombolytic efficacy of tissue plasminogen activator-loaded echogenic liposomes in a rabbit thrombus model. Research Support, Non-U.S. Gov't. Thrombosis research. Oct 2012;130(4):629-35. doi:10.1016/j.thromres.2011.11.010
22. Klegerman ME. Translational initiatives in thrombolytic therapy. Review. Frontiers of medicine. Mar 2017;11(1):1-19. doi:10.1007/s11684-017-0497-8
23. Thomas GR, Thibodeaux H, Bennett WF, et al. Optimized thrombolysis of cerebral clots with tissue-type plasminogen activator in a rabbit model of embolic stroke. Comparative Study. The Journal of pharmacology and experimental therapeutics. Jan 1993;264(1):67-73.
24. Alkan-Onyuksel H, Demos SM, Lanza GM, et al. Development of inherently echogenic liposomes as an ultrasonic contrast agent. J Pharm Sci. May 1996;85(5):486-90. doi:10.1021/js950407f
10.1021/js950407f [pii]
25. Demos SM, Onyuksel H, Gilbert J, et al. In vitro targeting of antibody-conjugated echogenic liposomes for site-specific ultrasonic image enhancement. J Pharm Sci. Feb 1997;86(2):167-71.
26. Cohen MG, E ET, Bluguermann J, et al. Transcutaneous ultrasound-facilitated coronary thrombolysis during acute myocardial infarction. American Journal of Cardiology. 2003;92:454-457.
27. Eggers J, Koch B, Meyer K, Konig I, Seidel G. Effect of ultrasound on thrombolysis of middle cerebral artery occlusion. Ann Neurol. Jun 2003;53(6):797-800.
28. Daffertshofer M, Hennerici M. Ultrasound in the treatment of ischaemic stroke. Lancet Neurology. 2003;2(5):283-290.
29. Koch S, Pohl P, Cobet U, Rainov NG. Ultrasound enhancement of liposome-mediated cell transfection is caused by cavitation effects. Ultrasound in Medicine and Biology. 2000;26(5):897-903.
30. Kim HJ, Greenleaf JF, Kinnick RR, Bronk JT, Bolander ME. Ultrasound-mediated transfection of mammalian cells. Human Gene Therapy. 1996;7(11):1339-1346.
31. Tachibana K, Tachibana S. The use of ultrasound for drug delivery. Echocardiography. 2001;18(4):323-328.
32. Kee PH, Moody MR, Huang SL, et al. Stabilizing Peri-Stent Restenosis Using a Novel Therapeutic Carrier. JACC Basic to translational science. Jan 2020;5(1):1-11. doi:10.1016/j.jacbts.2019.09.005
33. Klegerman ME, Naji AK, Haworth KJ, et al. Ultrasound-enhanced bevacizumab release from echogenic liposomes for inhibition of atheroma progression. Research Support, N.I.H., Extramural. Journal of liposome research. 2016;26(1):47-56. doi:10.3109/08982104.2015.1029494
34. Klegerman ME, Moody MR, Huang SL, et al. Demonstration of ultrasound-mediated therapeutic delivery of fibrin-targeted pioglitazone-loaded echogenic liposomes into the arterial bed for attenuation of peri-stent restenosis. Journal of drug targeting. Jan 2023;31(1):109-118. doi:10.1080/1061186X.2022.2110251
35. Birnbaum Y, Atar S, Luo H, Nagai T, Siegel RJ. Ultrasound has synergistic effects in vitro with tirofiban and heparin for thrombus dissolution. Thrombosis research. 1999;96(6):451-458.
36. Greenleaf WJ, Bolander ME, Sarkar G, Goldring MB, Greenleaf JF. Artificial cavitation nuclei significantly enhance acoustically induced cell transfection. Ultrasound in Medicine and Biology. 1998;24(4):587-595.
37. Zivin JA. tPA for Stroke: Story of a Controversial Drug. Oxford University Press; 2011.
38. Fan X, Qiu J, Yu Z, et al. A rat model of studying tissue-type plasminogen activator thrombolysis in ischemic stroke with diabetes. Stroke. Feb 2012;43(2):567-70. doi:10.1161/STROKEAHA.111.635250.
39. Tomkins AJ, Hood RJ, Levi CR, Spratt NJ. Tissue Plasminogen Activator for preclinical stroke research: Neither "rat" nor "human" dose mimics clinical recanalization in a carotid occlusion model. Sci Rep. Nov 2 2015;5:16026. doi:10.1038/srep16026.
2. Tsao CW, Aday AW, Almarzooq ZI, et al. Heart Disease and Stroke Statistics-2022 Update: A Report From the American Heart Association. Circulation. Feb 22 2022;145(8):e153-e639. doi:10.1161/CIR.0000000000001052
3. Tissue plasminogen activator for acute ischemic stroke. The National Institute of Neurological Disorders and Stroke rt-PA Stroke Study Group. Clinical Trial Multicenter Study Randomized Controlled Trial Research Support, U.S. Gov't, P.H.S. The New England journal of medicine. Dec 14 1995; 333(24): 1581-7. doi:10.1056/NEJM199512143332401
4. Collen D, Lijnen HR. Tissue-type plasminogen activator: a historical perspective and personal account. Historical Article Review. Journal of thrombosis and haemostasis : JTH. Apr 2004;2(4):541-6. doi:10.1111/j.1538-7933.2004.00645.x
5. Dundar Y, Hill R, Dickson R, Walley T. Comparative efficacy of thrombolytics in acute myocardial infarction: a systematic review. Comparative Study Meta-Analysis Review. QJM : monthly journal of the Association of Physicians. Feb 2003;96(2):103-13.
6. Bode C, Nordt TK, Peter K, Smalling RW, Runge MS, Kubler W. Patency trials with reteplase (r-PA): what do they tell us? Clinical Trial Comparative Study Randomized Controlled Trial Review. The American journal of cardiology. Dec 19 1996;78(12A):16-9.
7. Onundarson PT, Francis CW, Marder VJ. Depletion of plasminogen in vitro or during thrombolytic therapy limits fibrinolytic potential. Research Support, U.S. Gov't, P.H.S. The Journal of laboratory and clinical medicine. Jul 1992;120(1):120-8.
8. Sakharov DV, Rijken DC. Superficial accumulation of plasminogen during plasma clot lysis. Circulation. Oct 1 1995;92(7):1883-90.
9. Daffertshofer M, Gass A, Ringleb P, et al. Transcranial low-frequency ultrasound-mediated thrombolysis in brain ischemia: increased risk of hemorrhage with combined ultrasound and tissue plasminogen activator: results of a phase II clinical trial. Clinical Trial, Phase II Multicenter Study
Research Support, Non-U.S. Gov't. Stroke; a journal of cerebral circulation. Jul 2005;36(7):1441-6. doi:10.1161/01.STR.0000170707.86793.1a
10. Graham GD. Tissue plasminogen activator for acute ischemic stroke in clinical practice: a meta-analysis of safety data. Meta-Analysis Research Support, U.S. Gov't, P.H.S. Stroke; a journal of cerebral circulation. Dec 2003;34(12):2847-50. doi:10.1161/01.STR.0000101752.23813.C3
11. Katzan IL, Hammer MD, Hixson ED, Furlan AJ, Abou-Chebl A, Nadzam DM. Utilization of intravenous tissue plasminogen activator for acute ischemic stroke. Comparative Study. Archives of neurology. Mar 2004;61(3):346-50. doi:10.1001/archneur.61.3.346
12. Liang BA, Lew R, Zivin JA. Review of tissue plasminogen activator, ischemic stroke, and potential legal issues. Review. Archives of neurology. Nov 2008;65(11):1429-33. doi:10.1001/archneur.65.11.1429
13. Zaidat OO, Wolfe T, Hussain SI, et al. Interventional acute ischemic stroke therapy with intracranial self-expanding stent. Letter. Stroke; a journal of cerebral circulation. Aug 2008;39(8):2392-5. doi:10.1161/STROKEAHA.107.510966
14. Alexandrov AV, Molina CA, Grotta JC, et al. Ultrasound-enhanced systemic thrombolysis for acute ischemic stroke. Clinical Trial Clinical Trial, Phase II Multicenter Study Randomized Controlled Trial
Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S. The New England journal of medicine. Nov 18 2004;351(21):2170-8. doi:10.1056/NEJMoa041175
15. Huang S, Hamilton AJ, Tiukinhoy SD, et al. Liposomes as ultrasound imaging contrast agents and as ultrasound-sensitive drug delivery agents. Lectures. Cellular & molecular biology letters. 2002;7(2):233-5.
16. Huang SL. Liposomes in ultrasonic drug and gene delivery. Research Support, Non-U.S. Gov't
Review. Advanced drug delivery reviews. Jun 30 2008;60(10):1167-76. doi:10.1016/j.addr.2008.03.003
17. Tiukinhoy-Laing SD, Huang S, Klegerman M, Holland CK, McPherson DD. Ultrasound-facilitated thrombolysis using tissue-plasminogen activator-loaded echogenic liposomes. Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't. Thrombosis research. 2007;119(6):777-84. doi:10.1016/j.thromres.2006.06.009
18. Laing ST, Moody MR, Smulevitz B, et al. Doppler ultrasound enhances the thrombolytic activity of tissue plasminogen activator-loaded echogenic liposomes in vivo. Circulation. 2008;118(18):S643.
19. Moody M, Laing ST, Huang S, et al. Doppler ultrasound enhances the thrombolytic activity of tissue-plasminogen activator-loaded echogenic liposomes. Abstract. Circulation. October 16, 2007 2007;116(16):II-646.
20. Laing ST, Moody M, Smulevitz B, et al. Ultrasound-enhanced thrombolytic effect of tissue plasminogen activator-loaded echogenic liposomes in an in vivo rabbit aorta thrombus model--brief report. Research Support, N.I.H., Extramural. Arteriosclerosis, thrombosis, and vascular biology. Jun 2011;31(6):1357-9. doi:10.1161/ATVBAHA.111.225938
21. Laing ST, Moody MR, Kim H, et al. Thrombolytic efficacy of tissue plasminogen activator-loaded echogenic liposomes in a rabbit thrombus model. Research Support, Non-U.S. Gov't. Thrombosis research. Oct 2012;130(4):629-35. doi:10.1016/j.thromres.2011.11.010
22. Klegerman ME. Translational initiatives in thrombolytic therapy. Review. Frontiers of medicine. Mar 2017;11(1):1-19. doi:10.1007/s11684-017-0497-8
23. Thomas GR, Thibodeaux H, Bennett WF, et al. Optimized thrombolysis of cerebral clots with tissue-type plasminogen activator in a rabbit model of embolic stroke. Comparative Study. The Journal of pharmacology and experimental therapeutics. Jan 1993;264(1):67-73.
24. Alkan-Onyuksel H, Demos SM, Lanza GM, et al. Development of inherently echogenic liposomes as an ultrasonic contrast agent. J Pharm Sci. May 1996;85(5):486-90. doi:10.1021/js950407f
10.1021/js950407f [pii]
25. Demos SM, Onyuksel H, Gilbert J, et al. In vitro targeting of antibody-conjugated echogenic liposomes for site-specific ultrasonic image enhancement. J Pharm Sci. Feb 1997;86(2):167-71.
26. Cohen MG, E ET, Bluguermann J, et al. Transcutaneous ultrasound-facilitated coronary thrombolysis during acute myocardial infarction. American Journal of Cardiology. 2003;92:454-457.
27. Eggers J, Koch B, Meyer K, Konig I, Seidel G. Effect of ultrasound on thrombolysis of middle cerebral artery occlusion. Ann Neurol. Jun 2003;53(6):797-800.
28. Daffertshofer M, Hennerici M. Ultrasound in the treatment of ischaemic stroke. Lancet Neurology. 2003;2(5):283-290.
29. Koch S, Pohl P, Cobet U, Rainov NG. Ultrasound enhancement of liposome-mediated cell transfection is caused by cavitation effects. Ultrasound in Medicine and Biology. 2000;26(5):897-903.
30. Kim HJ, Greenleaf JF, Kinnick RR, Bronk JT, Bolander ME. Ultrasound-mediated transfection of mammalian cells. Human Gene Therapy. 1996;7(11):1339-1346.
31. Tachibana K, Tachibana S. The use of ultrasound for drug delivery. Echocardiography. 2001;18(4):323-328.
32. Kee PH, Moody MR, Huang SL, et al. Stabilizing Peri-Stent Restenosis Using a Novel Therapeutic Carrier. JACC Basic to translational science. Jan 2020;5(1):1-11. doi:10.1016/j.jacbts.2019.09.005
33. Klegerman ME, Naji AK, Haworth KJ, et al. Ultrasound-enhanced bevacizumab release from echogenic liposomes for inhibition of atheroma progression. Research Support, N.I.H., Extramural. Journal of liposome research. 2016;26(1):47-56. doi:10.3109/08982104.2015.1029494
34. Klegerman ME, Moody MR, Huang SL, et al. Demonstration of ultrasound-mediated therapeutic delivery of fibrin-targeted pioglitazone-loaded echogenic liposomes into the arterial bed for attenuation of peri-stent restenosis. Journal of drug targeting. Jan 2023;31(1):109-118. doi:10.1080/1061186X.2022.2110251
35. Birnbaum Y, Atar S, Luo H, Nagai T, Siegel RJ. Ultrasound has synergistic effects in vitro with tirofiban and heparin for thrombus dissolution. Thrombosis research. 1999;96(6):451-458.
36. Greenleaf WJ, Bolander ME, Sarkar G, Goldring MB, Greenleaf JF. Artificial cavitation nuclei significantly enhance acoustically induced cell transfection. Ultrasound in Medicine and Biology. 1998;24(4):587-595.
37. Zivin JA. tPA for Stroke: Story of a Controversial Drug. Oxford University Press; 2011.
38. Fan X, Qiu J, Yu Z, et al. A rat model of studying tissue-type plasminogen activator thrombolysis in ischemic stroke with diabetes. Stroke. Feb 2012;43(2):567-70. doi:10.1161/STROKEAHA.111.635250.
39. Tomkins AJ, Hood RJ, Levi CR, Spratt NJ. Tissue Plasminogen Activator for preclinical stroke research: Neither "rat" nor "human" dose mimics clinical recanalization in a carotid occlusion model. Sci Rep. Nov 2 2015;5:16026. doi:10.1038/srep16026.