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

Abstract

<jats:title>Summary</jats:title><jats:p>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.</jats:p>

Real‐time tracking of fibrinolysis under constant wall shear and various pulsatile flows in an in‐vitro thrombolysis model

“…One limitation of the RT‐FluFF assay remains to be the in‐vitro nature of the setup as it cannot offer information on drug toxicity and pharmacokinetics despite perfusion of human blood or plasma. The recent advancements of intravital microscopy make it possible for real‐time imaging of thrombolysis in animals 47,48 . Since animals, especially small rodents, bear distinct physiology and hemodynamics to that of humans, the RT‐FluFF assay offers complementary drug testing results in the preclinical phase in a more human physiologically similar system without requiring live animal experimentation.…”

Experimental Models to Study Skin Wound Healing with a Focus on Angiogenesis

“…Since then, microcirculation and tissue engineering research have frequently used the mouse dorsal skinfold chamber [19]. Microcirculatory studies focused on inflammation [20,21], thrombogenesis [22,23], thrombolysis [24,25], angiogenesis of tumors [26][27][28], endometriosis [29,30], biomaterials [31,32] and flap perfusion [33,34]. The number of studies using the dorsal skinfold chamber model for different approaches proves the dorsal skinfold chamber to be one of the most important in vivo models for repetitive microcirculation assessment.…”

In vivo analysis of noise dependent activation of white blood cells and microvascular dysfunction in mice

“…The dorsal skinfold chamber facilitates an unobstructed insight to the intramuscular vessel network through exposure of the panniculus carnosus muscle in mice. Moreover, application of fluorophores allows the visualization blood plasma [8] and specific blood cells, including, leukocytes [9] , and thrombocytes [10] .…”