Department of Neurology, Saitama Medical University International Medical Center, Saitama, 350-1298, Japan.
J Thromb Thrombolysis. 2012 Aug;34(2):193-8. doi: 10.1007/s11239-012-0703-0.
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 μm(2)) than in the control group (358 ± 256 μm(2)) or clopidogrel (1 mg/kg) group (355 ± 57 μm(2)). 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.
抗血小板药物已通过测量血小板聚集来进行评估，但体内研究较少。本研究旨在通过活体荧光显微镜观察抗血小板药物（氯吡格雷）对小鼠激光诱导血栓形成过程的影响。使用氯醛水麻醉 C57BL/6J 小鼠（n = 19）。用头部固定器固定每只老鼠的头部，并在前额区域制作颅窗。通过静脉内给予羧基荧光素二乙酸琥珀酰亚胺酯使血小板在体内标记。氯吡格雷（1 mg/kg，n = 6；10 mg/kg，n = 6）在实验前口服给药 2 天。另外 7 只小鼠作为对照。激光照射（1000 mA，9.8 mW，二极管泵浦固态（DPSS）激光 532nm）持续 4s 于软脑膜动脉，以诱导血栓形成。荧光显微镜下连续观察标记的血小板和血栓。记录 30 分钟后的血栓面积，并确定完全闭塞率。激光照射软脑膜动脉后，氯吡格雷（10mg/kg）组的完全闭塞率（16%，25 个血管中有 4 个）明显低于对照组（60%，20 个血管中有 12 个）或氯吡格雷（1mg/kg）组（55%，20 个血管中有 11 个）。激光照射 30 分钟后血小板血栓面积在氯吡格雷（10mg/kg）组（209 ± 128μm(2))明显小于对照组（358 ± 256μm(2))或氯吡格雷（1mg/kg）组（355 ± 57μm(2))。我们开发的仪器通过对小动物脑表面血管内皮细胞的损伤，方便地在不损伤血管外组织的情况下诱导血栓形成。氯吡格雷以剂量依赖性方式显著抑制小鼠软脑膜动脉的激光诱导血栓形成。
