From the Department of Epidemiology, Atherothrombosis, and Imaging, Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain (R.C., J.M.G.-G., V.S., C.S.-R., P.M.-S., A.H., V.A.); Institute for Cardiovascular Prevention, Ludwig-Maximilians-University Munich and DZHK (German Centre for Cardiovascular Research), partner site Munich Heart Alliance, Munich, Germany (R.T.A.M., C.W., O.S.); Cardiovascular Research Institute Maastricht, Maastricht, The Netherlands (R.T.A.M., C.W.); and Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands (O.S.).
Circ Res. 2014 Feb 28;114(5):770-9. doi: 10.1161/CIRCRESAHA.114.302590. Epub 2013 Dec 23.
The inflammatory processes that initiate and propagate atherosclerosis remain poorly understood, largely because defining the intravascular behavior of immune cells has been technically challenging. Respiratory and pulsatile movements have hampered in vivo visualization of leukocyte accumulation in athero-prone arteries at resolutions achieved in other tissues.
To establish and to validate a method that allows high-resolution imaging of inflammatory leukocytes and platelets within the carotid artery of atherosusceptible mice in vivo.
We have devised a procedure to stabilize the mouse carotid artery mechanically without altering blood dynamics, which dramatically enhances temporal and spatial resolutions using high-speed intravital microscopy in multiple channels of fluorescence. By applying this methodology at different stages of disease progression in atherosusceptible mice, we first validated our approach by assessing the recruitment kinetics of various leukocyte subsets and platelets in athero-prone segments of the carotid artery. The high temporal and spatial resolution allowed the dissection of both the dynamic polarization of and the formation of subcellular domains within adhered leukocytes. We further demonstrate that the secondary capture of activated platelets on the plaque is predominantly mediated by neutrophils. Finally, we couple this procedure with triggered 2-photon microscopy to visualize the 3-dimensional movement of leukocytes in intimate contact with the arterial lumen.
The improved imaging of diseased arteries at subcellular resolution presented here should help resolve many outstanding questions in atherosclerosis and other arterial disorders.
引发和传播动脉粥样硬化的炎症过程仍未被充分理解，主要是因为定义血管内免疫细胞的行为在技术上具有挑战性。呼吸和脉动运动阻碍了在其他组织中实现的分辨率下对动脉粥样易损部位中白细胞积累的体内可视化。
建立并验证一种方法，该方法允许在体内对易感动脉粥样硬化的小鼠颈动脉内的炎症性白细胞和血小板进行高分辨率成像。
我们设计了一种机械稳定小鼠颈动脉的程序，而不会改变血液动力学，这极大地提高了使用高速活体显微镜在多个荧光通道中的时间和空间分辨率。通过在易感小鼠疾病进展的不同阶段应用这种方法，我们首先通过评估各种白细胞亚群和血小板在颈动脉易损部位的募集动力学来验证我们的方法。高时间和空间分辨率允许对附着的白细胞内的动态极化和亚细胞结构域的形成进行剖析。我们进一步证明，斑块上活化血小板的二次捕获主要是由中性粒细胞介导的。最后，我们将此程序与触发式双光子显微镜相结合，以可视化与动脉腔紧密接触的白细胞的 3 维运动。
这里呈现的亚细胞分辨率下对病变动脉的改进成像应该有助于解决动脉粥样硬化和其他动脉疾病中的许多悬而未决的问题。
