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Thromb Haemost 2012; 107(05): 962-971
DOI: 10.1160/TH11-10-0705
Animal Models
Schattauer GmbH

The mouse dorsal skinfold chamber as a model for the study of thrombolysis by intravital microscopy

Yacine Boulaftali  
1   University Paris Diderot, Sorbonne Paris Cité, Inserm UMR698, Paris, France
2   AP-HP Hospital Bichat Claude Bernard, Paris, France
,
Lamia Lamrani  
1   University Paris Diderot, Sorbonne Paris Cité, Inserm UMR698, Paris, France
2   AP-HP Hospital Bichat Claude Bernard, Paris, France
,
Marie-Catherine Rouzaud
1   University Paris Diderot, Sorbonne Paris Cité, Inserm UMR698, Paris, France
,
Stéphane Loyau
1   University Paris Diderot, Sorbonne Paris Cité, Inserm UMR698, Paris, France
2   AP-HP Hospital Bichat Claude Bernard, Paris, France
,
Martine Jandrot-Perrus
1   University Paris Diderot, Sorbonne Paris Cité, Inserm UMR698, Paris, France
2   AP-HP Hospital Bichat Claude Bernard, Paris, France
,
Marie-Christine Bouton
1   University Paris Diderot, Sorbonne Paris Cité, Inserm UMR698, Paris, France
2   AP-HP Hospital Bichat Claude Bernard, Paris, France
,
Benoît Ho-Tin-Noé
1   University Paris Diderot, Sorbonne Paris Cité, Inserm UMR698, Paris, France
2   AP-HP Hospital Bichat Claude Bernard, Paris, France
› Author Affiliations Financial support: This study was supported by Institut National de la Santé et de la Recherche Médicale and by grants from La Fondation pour la Recherche Médicale and from La Fondation de France (grant n°2009002497).
Further Information

Publication History

Received: 13 October 2011

Accepted after major revision: 24 January 2012

Publication Date:
25 November 2017 (online)

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Summary

Although intravital microscopy models of thrombosis in mice have contributed to dissect the mechanisms of thrombus formation and stability, they have not been well adapted to study long-term evolution of occlusive thrombi. Here, we assessed the suitability of the dorsal skinfold chamber (DSC) for the study of thrombolysis and testing of thrombolytic agents by intravital microscopy. We show that induction of FeCl3-induced occlusive thrombosis is achievable in microvessels of DSCs, and that thrombi formed in DSCs can be visualised by intravital microscopy using brightfield transmitted light, or fluorescent staining of thrombus components such as fibrinogen, platelets, leukocytes, and von Willebrand factor. Direct application of control saline or recombinant tissue-plasminogen activator (rtPA) to FeCl3-produced thrombi in DSCs did not affect thrombus size or induce recanalisation. However, in the presence of hirudin, rtPA treatment caused a rapid dose-dependent lysis of occlusive thrombi, resulting in recanalisation within 1 hour after treatment. Skin haemorrhage originating from vessels located inside and outside the FeCl3-injured area was also observed in DSCs of rtPA-treated mice. We further show that rtPA-induced thrombolysis was enhanced in plasminogen activator inhibitor-1-deficient (PAI-1−/−) mice, and dropped considerably as the time between occlusion and treatment application increased. Together, our results show that by allowing visualization and measurement of thrombus lysis and potential bleeding complications of thrombolytic treatments, the DSC provides a model for studying endogenous fibrinolysis and for first-line screening of thrombolytic agents. Furthermore, using this system, we found that thrombin and clot aging impair the thrombolytic action of rtPA towards FeCl3-produced thrombi.

Keywords

Dorsal skinfold chamber - intravital microscopy - thrombolysis - thrombosis - tPA

Y.B. and L.L. contributed equally to this study.


 

  • References

  • 1 Bizzozero G. Ueber einen neuen Formbestandteil des Blutes und dessen Rolle bei der Thrombose und Blutgerinnung. Archiv für pathologische Anatomie und Physiologie und für klinische Medicin 1882; 90: 261-332.
    CrossrefPubMedGoogle Scholar
  • 2 Chauhan AK, Motto DG, Lamb CB. et al. Systemic antithrombotic effects of ADAMTS13. J Exp Med 2006; 203: 767-76.
    CrossrefPubMedGoogle Scholar
  • 3 Boulaftali Y, Adam F, Venisse L. et al. Anticoagulant and antithrombotic properties of platelet protease nexin-1. Blood 2010; 115: 97-106.
    CrossrefPubMedGoogle Scholar
  • 4 Cambien B, Bergmeier W, Saffaripour S. et al. Antithrombotic activity of TNF-alpha. J Clin Invest 2003; 112: 1589-1596.
    CrossrefPubMedGoogle Scholar
  • 5 Diener JL, Daniel Lagasse HA, Duerschmied D. et al. Inhibition of von Willebrand factor-mediated platelet activation and thrombosis by the anti-von Willebrand factor A1-domain aptamer ARC1779. J Thromb Haemost 2009; 07: 1155-1162.
    CrossrefPubMedGoogle Scholar
  • 6 Ni H, Denis CV, Subbarao S. et al. Persistence of platelet thrombus formation in arterioles of mice lacking both von Willebrand factor and fibrinogen. J Clin Invest 2000; 106: 385-392.
    CrossrefPubMedGoogle Scholar
  • 7 Ni H, Papalia JM, Degen JL. et al. Control of thrombus embolization and fibronectin internalization by integrin alpha IIb beta 3 engagement of the fibrinogen gamma chain. Blood 2003; 102: 3609-3614.
    CrossrefPubMedGoogle Scholar
  • 8 Ni H, Ramakrishnan V, Ruggeri ZM. et al. Increased thrombogenesis and embolus formation in mice lacking glycoprotein V. Blood 2001; 98: 368-373.
    CrossrefPubMedGoogle Scholar
  • 9 Wang YX, da Cunha V, Vincelette J. et al. A novel inhibitor of activated thrombin activatable fibrinolysis inhibitor (TAFIa) - part II: enhancement of both exogenous and endogenous fibrinolysis in animal models of thrombosis. Thromb Haemost 2007; 97: 54-61.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 10 Wadanoli M, Sako D, Shaw GD. et al. The von Willebrand factor antagonist (GPG-290) prevents coronary thrombosis without prolongation of bleeding time. Thromb Haemost 2007; 98: 397-405.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 11 Zhu Y, Carmeliet P, Fay WP. Plasminogen activator inhibitor-1 is a major determinant of arterial thrombolysis resistance. Circulation 1999; 99: 3050-3055.
    CrossrefPubMedGoogle Scholar
  • 12 Orbe J, Barrenetxe J, Rodriguez JA. et al. Matrix Metalloproteinase-10 Effectively Reduces Infarct Size in Experimental Stroke by Enhancing Fibrinolysis via a Thrombin-Activatable Fibrinolysis Inhibitor-Mediated Mechanism. Circulation 2011; 124: 2909-2919.
    CrossrefPubMedGoogle Scholar
  • 13 Roussel BD, Mysiorek C, Rouhiainen A. et al. HMGB-1 promotes fibrinolysis and reduces neurotoxicity mediated by tissue plasminogen activator. J Cell Sci 2011; 124: 2070-2076.
    CrossrefPubMedGoogle Scholar
  • 14 Koehl GE, Gaumann A, Geissler EK. Intravital microscopy of tumor angiogenesis and regression in the dorsal skin fold chamber: mechanistic insights and preclinical testing of therapeutic strategies. Clin Exp Metastasis 2009; 26: 329-344.
    CrossrefPubMedGoogle Scholar
  • 15 Harder Y, Amon M, Erni D. et al. Evolution of ischemic tissue injury in a random pattern flap: a new mouse model using intravital microscopy. J Surg Res 2004; 121: 197-205.
    CrossrefPubMedGoogle Scholar
  • 16 Nolte D, Menger MD, Messmer K. Microcirculatory models of ischaemia-reperfusion in skin and striated muscle. Int J Microcirc Clin Exp 1995; 15 (Suppl. 01) 9-16.
    CrossrefPubMedGoogle Scholar
  • 17 Lehr HA, Leunig M, Menger MD. et al. Dorsal skinfold chamber technique for intravital microscopy in nude mice. Am J Pathol 1993; 143: 1055-1062.
    PubMedGoogle Scholar
  • 18 Eckly A, Hechler B, Freund M. et al. Mechanisms underlying FeCl3-induced arterial thrombosis. J Thromb Haemost 2011; 09: 779-789.
    CrossrefPubMedGoogle Scholar
  • 19 Broersma RJ, Kutcher LW, Heminger E F. The effect of thrombin inhibit ion in a rat arterial thrombosis model. Thromb Res 1991; 64: 405-412.
    CrossrefPubMedGoogle Scholar
  • 20 Kuijpers MJ, Munnix IC, Cosemans JM. et al. Key role of platelet procoagulant activity in tissue factor-and collagen-dependent thrombus formation in arterioles and venules in vivo differential sensitivity to thrombin inhibition. Microcirculation 2008; 15: 269-282.
    CrossrefPubMedGoogle Scholar
  • 21 Wang X, Cheng Q, Xu L. et al. Effects of factor IX or factor XI deficiency on ferric chloride-induced carotid artery occlusion in mice. J Thromb Haemost 2005; 03: 695-702.
    CrossrefPubMedGoogle Scholar
  • 22 Day SM, Reeve JL, Pedersen B. et al. Macrovascular thrombosis is driven by tissue factor derived primarily from the blood vessel wall. Blood 2005; 105: 192-198.
    CrossrefPubMedGoogle Scholar
  • 23 Grines CL, Serruys P, O'Neill WW. Fibrinolytic therapy: is it a treatment of the past?. Circulation 2003; 107: 2538-2542.
    CrossrefPubMedGoogle Scholar
  • 24 Kilic E, Hermann DM, Hossmann KA. Recombinant tissue-plasminogen activator-induced thrombolysis after cerebral thromboembolism in mice. Acta Neuropathol 2000; 99: 219-222.
    CrossrefPubMedGoogle Scholar
  • 25 Orset C, Macrez R, Young AR. et al. Mouse model of in situ thromboembolic stroke and reperfusion. Stroke 2007; 38: 2771-2778.
    CrossrefPubMedGoogle Scholar
  • 26 Jang IK, Gold HK, Ziskind AA. et al. Differential sensitivity of erythrocyte-rich and platelet-rich arterial thrombi to lysis with recombinant tissue-type plasminogen activator. A possible explanation for resistance to coronary thrombolysis. Circulation 1989; 79: 920-928.
    CrossrefPubMedGoogle Scholar
  • 27 Mazighi M, Serfaty JM, Labreuche J. et al. Comparison of intravenous alteplase with a combined intravenous-endovascular approach in patients with stroke and confirmed arterial occlusion (RECANALISE study): a prospective cohort study. Lancet Neurol 2009; 08: 802-809.
    CrossrefPubMedGoogle Scholar
  • 28 Ouriel K. Current status of thrombolysis for peripheral arterial occlusive disease. Ann Vasc Surg 2002; 16: 797-804.
    CrossrefPubMedGoogle Scholar
  • 29 Kim HS, Preece SR, Black JH. et al. Safety of catheter-directed thrombolysis for deep venous thrombosis in cancer patients. J Vasc Surg 2008; 47: 388-394.
    CrossrefPubMedGoogle Scholar
  • 30 Mewissen MW, Seabrook GR, Meissner MH. et al. Catheter-directed thrombolysis for lower extremity deep venous thrombosis: report of a national multicenter registry. Radiology 1999; 211: 39-49.
    CrossrefPubMedGoogle Scholar
  • 31 Kuo WT, van den Bosch MA, Hofmann LV. et al. Catheter-directed embolectomy, fragmentation, and thrombolysis for the treatment of massive pulmonary embolism after failure of systemic thrombolysis. Chest 2008; 134: 250-254.
    CrossrefPubMedGoogle Scholar
  • 32 Kuo WT, Gould MK, Louie JD. et al. Catheter-directed therapy for the treatment of massive pulmonary embolism: systematic review and meta-analysis of modern techniques. J Vasc Interv Radiol 2009; 20: 1431-1440.
    CrossrefPubMedGoogle Scholar
  • 33 Korninger C, Collen D. Studies on the specific fibrinolytic effect of human extrinsic (tissue-type) plasminogen activator in human blood and in various animal species in vitro. Thromb Haemost 1981; 46: 561-565.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 34 Lijnen HR, van Hoef B, Beelen V. et al. Characterization of the murine plasma fibrinolytic system. Eur J Biochem 1994; 224: 863-871.
    CrossrefPubMedGoogle Scholar
  • 35 Lapergue B, Moreno JA, Dang BQ. et al. Protective effect of high-density lipoprotein-based therapy in a model of embolic stroke. Stroke 2010; 41: 1536-1542.
    CrossrefPubMedGoogle Scholar
  • 36 Atochin DN, Murciano JC, Gursoy-Ozdemir Y. et al. Mouse model of microembolic stroke and reperfusion. Stroke 2004; 35: 2177-2182.
    CrossrefPubMedGoogle Scholar
  • 37 Dubois C, Panicot-Dubois L, Merrill-Skoloff G. et al. Glycoprotein VI-dependent and -independent pathways of thrombus formation in vivo. Blood 2006; 107: 3902-3906.
    CrossrefPubMedGoogle Scholar
  • 38 Boulaftali Y, Ho-Tin-Noe B, Pena A. et al. Platelet protease nexin-1, a serpin that strongly influences fibrinolysis and thrombolysis. Circulation 2011; 123: 1326-1334.
    CrossrefPubMedGoogle Scholar
  • 39 Fay WP, Eitzman DT, Shapiro AD. et al. Platelets inhibit fibrinolysis in vitro by both plasminogen activator inhibitor-1-dependent and -independent mechanisms. Blood 1994; 83: 351-356.
    PubMedGoogle Scholar
  • 40 Francis CW, Marder VJ. Rapid formation of large molecular weight alphapolymers in cross-linked fibrin induced by high factor XIII concentrations. Role of platelet factor XIII. J Clin Invest 1987; 80: 1459-1465.
    PubMedGoogle Scholar
  • 41 McDonagh J, McDonagh Jr. RP, Delage JM. et al. Factor XIII in human plasma and platelets. J Clin Invest 1969; 48: 940-946.
    CrossrefPubMedGoogle Scholar
  • 42 Kunitada S, FitzGerald GA, Fitzgerald DJ. Inhibition of clot lysis and decreased binding of tissue-type plasminogen activator as a consequence of clot retraction. Blood 1992; 79: 1420-1427.
    PubMedGoogle Scholar
  • 43 Zaidat OO, Suarez JI, Sunshine JL. et al. Thrombolytic therapy of acute ischemic stroke: correlation of angiographic recanalization with clinical outcome. AJNR Am J Neuroradiol 2005; 26: 880-884.
    PubMedGoogle Scholar
  • 44 Lees KR, Bluhmki E, von Kummer R. et al. Time to treatment with intravenous alteplase and outcome in stroke: an updated pooled analysis of ECASS, ATLANTIS, NINDS, and EPITHET trials. Lancet 2010; 375: 1695-1703.
    CrossrefPubMedGoogle Scholar
  • 45 Westerhout CM, Bonnefoy E, Welsh RC. et al. The influence of time from symptom onset and reperfusion strategy on 1-year survival in ST-elevation myocardial infarction: a pooled analysis of an early fibrinolytic strategy versus primary percutaneous coronary intervention from CAPTIM and WEST. Am Heart J 2011; 161: 283-290.
    CrossrefPubMedGoogle Scholar
  • 46 Steg PG, Bonnefoy E, Chabaud S. et al. Impact of time to treatment on mortality after prehospital fibrinolysis or primary angioplasty: data from the CAPTIM randomized clinical trial. Circulation 2003; 108: 2851-2856.
    CrossrefPubMedGoogle Scholar
  • 47 Kennedy JW. Optimal management of acute myocardial infarction requires early and complete reperfusion. Circulation 1995; 91: 1905-1907.
    CrossrefPubMedGoogle Scholar