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Laser-induced thrombus formation in mouse brain microvasculature: effect of clopidogrel

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Abstract

Antiplatelet drugs have been evaluated by measuring platelet aggregation ex vivo, but in vivo studies were scanty. The purpose of this study was to observe the effects of an antiplatelet agent (clopidogrel) on the process of laser-induced thrombus formation in mice using intravital fluorescence microscopy. C57 BL/6J mice (n = 19) were anesthetized using chloral hydrate. The head of each mouse was fixed with a head holder, and a cranial window was made in the parietal region. Platelets were labeled in vivo by intravenous administration of carboxyfluorescein diacetate succinimidyl ester. Clopidogrel (1 mg/kg, n = 6; 10 mg/kg, n = 6) was administered orally for 2 days before the experiment. Another seven mice were used as controls. Laser irradiation (1,000 mA, 9.8 mW, diode-pumped solid-state (DPSS) laser 532 nm) was directed for 4 s at pial arteries to induce thrombus formation. Labeled platelets and thrombus were observed continuously under fluorescence microscopy. We recorded the area of thrombus after 30 min and determined the complete occlusion rate. After laser irradiation to the pial artery, complete occlusion rate was significantly lower in the clopidogrel (10 mg/kg) group (16%, 4/25 vessels) than in the control group (60%, 12/20 vessels) or clopidogrel (1 mg/kg) group (55%, 11/20 vessels). Area of platelet thrombus at 30 min after laser irradiation was significantly smaller in the clopidogrel (10 mg/kg) group (209 ± 128 μm2) than in the control group (358 ± 256 μm2) or clopidogrel (1 mg/kg) group (355 ± 57 μm2). The apparatus which we developed is convenient for inducing thrombus formation by causing endothelial cell damage to the brain surface vasculature in small animals without damage of extravascular tissue. Clopidogrel significantly inhibited laser-induced thrombus formation in pial arteries of mice in a dose-dependent manner.

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Acknowledgments

The authors wish to thank Professor Masumi Akita and Kayoko Tanaka from the Division of Morphological Science, Biomedical Research Center, Saitama Medical University for their cooperation in preparing electron micrographs of mouse pial arteries.

Conflicts of interest

NT has received payment for development of educational presentations from Mitsubishi Tanabe Pharma, Pfizer Japan, Sanofi-Aventis, and Otsuka Pharmaceutical. TF, KH, HM and MH declare that they have no conflicts of interest.

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Authors and Affiliations

  1. Department of Neurology, Saitama Medical University International Medical Center, Saitama, 350-1298, Japan

    Takuya Fukuoka, Kimihiko Hattori, Hajime Maruyama, Makiko Hirayama & Norio Tanahashi

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  1. Takuya Fukuoka
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  2. Kimihiko Hattori
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  3. Hajime Maruyama
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  4. Makiko Hirayama
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  5. Norio Tanahashi
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Corresponding author

Correspondence to Takuya Fukuoka.

Additional information

This study was supported by Grant from Ochiai memorial award 2011.

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Fukuoka, T., Hattori, K., Maruyama, H. et al. Laser-induced thrombus formation in mouse brain microvasculature: effect of clopidogrel. J Thromb Thrombolysis 34, 193–198 (2012). https://doi.org/10.1007/s11239-012-0703-0

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