A New Model for Quantitative In Vivo Microscopic Analysis of Thrombus Formation and Vascular Recanalisation: The Ear of the Hairless (hr/hr) Mouse

Abstract

SummaryThe alteration of rheological blood properties as well as deterioration of vascular perfusion conditions and cell-cell interactions are major determinants of thrombus formation. Herein, we present an experimental model which allows for quantitative in vivo microscopic analysis of these determinants during both thrombus formation and vascular recanalisation. The model does not require surgical preparation procedures, and enables for repeated analysis of identical microvessels over time periods of days or months, respectively. After i.v. administration of FITC-dextran thrombus formation was induced photochemically by light exposure to individual arterioles and venules of the ear of ten anaesthetised hairless mice. In venules, epiillumination induced rapid thrombus formation with first platelet deposition after 0.59 ± 0.04 min and complete vessel occlusion within 7.48 ±1.31 min. After a 24-h time period, 75% of the thrombosed venules were found recanalised. Marked leukocyte-endothelial cell interaction in those venules indicated persistent endothelial cell activation and/or injury, even after an observation period of 7 days. In arterioles, epi-illumination provoked vasomotion, while thrombus formation was significantly (p <0.05) delayed with first platelet deposition after 2.32 ± 0.22 min and complete vessel occlusion within 20.07 ±3.84 min. Strikingly, only one of the investigated arterioles was found recanalised after 24 h, which, however, did not show leukocyte-endothelial cell interaction. Heparin (300 U/kg, i.v.) effectively counteracted the process of thrombus formation in this model, including both first platelet deposition and vessel occlusion. We conclude that the model of the ear of the hairless mouse allows for distinct in vivo analysis of arteriolar and venular thrombus formation/ recanalisation, and, thus, represents an interesting tool for the study of novel antithrombotic and thrombolytic strategies, respectively.

A novel model of occlusive thrombus formation in mice

“…It is known that thrombus formation is prevented by the antithrombogenic function of endothelium and that the disruption of endothelial integrity is one of the pathogenesis of thrombus formation. 4,6,7,15 We showed that the GSL-1 binding and expression of vWF and eNOS as histological makers of endothelial cells were markedly decreased in endothelium that was accompanied by thrombus formation in ligation-cuff-treated arteries. Thus, the combination of ligation and cuff placement induced the damage of endothelia, which led to the exposure of the subendothelial matrix.…”

“…In most mouse models of thrombus formation, thrombi were rapidly generated from within a few seconds to less than 1 h. [4][5][6]25,26 These models by means of acute and dramatic injury of endothelium appear not to be suitable for investigating the course and mechanism of endothelium damage or injury, since it is too acute to detect the pathophysiological changes of endothelium after stimuli. This model is likely to be of particular value in studying the relationship between thrombosis formation and the alteration of endothelial cell morphology and function in detail.…”

“…Several models of murine thrombosis by vascular injury have been reported using mechanical, 3 electrical, 4 FeCl3, 5 photochemical 6 and laser-induced injuries. 7,8 The development of thrombi induced by most of these methods are very acute, occurring from within a few seconds to 1 h. However, a model in which occlusive thrombus is formed gradually in the carotid artery has not been reported.…”

Thrombus formation in vivo

“…Oude Egbrink et al were among the first to combine experimental thrombosis, induced by mechanical puncture with glass micropipettes, with intravital videomicroscopy in a living animal (23). Others applied experimental thrombosis with in vivo microscopic analysis (24,25), adopting the methods used for leukocyte rolling. Photochemical injury of microvessels in the mouse ear allowed analysis of the kinetics of platelet accumulation and vessel occlusion, leading to the observation that hirudin inhibited thrombus formation and promoted vessel recanalization (26).…”

In Vivo Models for the Evaluation of Antithrombotics and Thrombolytics

“…A model has been described in which FITC-dextran is administered intravenously to mice. Thrombus formation is induced upon exposure of the microvessels of the ear to the light of a mercury lamp (excitation wavelength of 450-490 nm) (97). The endothelial damage induced in this model is believed to be a result of the generation of singlet molecular oxygen produced by energy transfer from the excited dye (98).…”