Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati College of Medicine; Division of Molecular Cardiovascular Biology, Cincinnati Children's Hospital Medical Center.
Confocal Imaging Core, Cincinnati Children's Hospital Medical Center.
J Vis Exp. 2021 May 16(171). doi: 10.3791/62023.
Cardiovascular disease is the most prevalent cause of mortality worldwide and is often marked by heightened cardiac fibrosis that can lead to increased ventricular stiffness with altered cardiac function. This increase in cardiac ventricular fibrosis is due to activation of resident fibroblasts, although how these cells operate within the 3-dimensional (3-D) heart, at baseline or after activation, is not well understood. To examine how fibroblasts contribute to heart disease and their dynamics in the 3-D heart, a refined CLARITY-based tissue clearing and imaging method was developed that shows fluorescently labeled cardiac fibroblasts within the entire mouse heart. Tissue resident fibroblasts were genetically labeled using Rosa26-loxP-eGFP florescent reporter mice crossed with the cardiac fibroblast expressing Tcf21-MerCreMer knock-in line. This technique was used to observe fibroblast localization dynamics throughout the entire adult left ventricle in healthy mice and in fibrotic mouse models of heart disease. Interestingly, in one injury model, unique patterns of cardiac fibroblasts were observed in the injured mouse heart that followed bands of wrapped fibers in the contractile direction. In ischemic injury models, fibroblast death occurred, followed by repopulation from the infarct border zone. Collectively, this refined cardiac tissue clarifying technique and digitized imaging system allows for 3-D visualization of cardiac fibroblasts in the heart without the limitations of antibody penetration failure or previous issues surrounding lost fluorescence due to tissue processing.
心血管疾病是全球最常见的死亡原因，通常以心肌纤维化加剧为特征，这可能导致心室僵硬，心脏功能改变。心肌纤维化的增加是由于驻留成纤维细胞的激活，尽管这些细胞在 3 维（3-D）心脏中的运作方式，无论是在基础状态还是在激活后，都还不太清楚。为了研究成纤维细胞如何导致心脏病以及它们在 3-D 心脏中的动态变化，开发了一种改良的基于 CLARITY 的组织透明化和成像方法，该方法可在整个小鼠心脏中显示荧光标记的心肌成纤维细胞。使用 Rosa26-loxP-eGFP 荧光报告小鼠与表达 Tcf21-MerCreMer 的心脏成纤维细胞敲入系杂交，对组织驻留成纤维细胞进行基因标记。该技术用于观察健康小鼠和心肌疾病纤维化模型中整个左心室的成纤维细胞定位动力学。有趣的是，在一种损伤模型中，在损伤的小鼠心脏中观察到了心肌成纤维细胞的独特模式，这些模式沿着收缩方向的缠绕纤维带分布。在缺血性损伤模型中，成纤维细胞死亡，随后从梗死边缘区重新出现。总的来说，这种改良的心脏组织透明化技术和数字化成像系统允许在不限制抗体渗透失败或由于组织处理导致荧光丢失的情况下，对心脏中的心肌成纤维细胞进行 3-D 可视化。
