In situ three-dimensional reconstruction of mouse heart sympathetic innervation by two-photon excitation fluorescence imaging.
In situ three-dimensional reconstruction of mouse heart sympathetic innervation by two-photon excitation fluorescence imaging.
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DOI：
10.1016/j.jneumeth.2013.09.005
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发表时间：
2014-01-15
影响因子：
3
通讯作者：
Rubart, Michael
中科院分区：
文献类型：
--
作者：
Freeman, Kim;Tao, Wen;Sun, Hongli;Soonpaa, Mark H.;Rubart, Michael
关键词：
哺乳动物心脏中的交感神经接线仍然在很大程度上未被探索。解析心脏交感神经网络的接线图将有助于建立心神经耦合的结构基础。我们使用双光子激发荧光显微镜，结合计算机辅助的3-D跟踪算法，映射局部交感神经回路在活的心脏从成年转基因小鼠表达增强型绿色荧光蛋白（EGFP）在外周肾上腺素能神经元。定量共定位分析证实，心肌内的EGFP分布概括了解剖的交感神经乔木。在未受伤心脏的左心室心外膜下，交感神经网络由多个亚动脉组成，表现出可变的分支和环路拓扑结构。轴突分支没有相互重叠在各自的父母subarbor也没有与附属subarbor的神经突。2周龄心肌梗死边缘区的交感神经网络的特点是实质性的重新布线，其中包括空间异质性损失和增益的交感神经纤维和形成多个，主要是嵌套，轴突环路的广泛变化的周长和几何形状。与可能涉及组织变形和跨切片配准不确定性的机械组织切片方法相比，我们的方法保留了结构的连续性，这允许在距离上追踪神经突，从而能够导出心脏交感神经的三维和拓扑形态。我们的试验应该是通用的解开交感神经轴突间距在发展和疾病的机制。
Sympathetic nerve wiring in the mammalian heart has remained largely unexplored. Resolving the wiring diagram of the cardiac sympathetic network would help establish the structural underpinnings of neurocardiac coupling. We used two-photon excitation fluorescence microscopy, combined with a computer-assisted 3-D tracking algorithm, to map the local sympathetic circuits in living hearts from adult transgenic mice expressing enhanced green fluorescent protein (EGFP) in peripheral adrenergic neurons. Quantitative co-localization analyses confirmed that the intramyocardial EGFP distribution recapitulated the anatomy of the sympathetic arbor. In the left ventricular subepicardium of the uninjured heart, the sympathetic network was composed of multiple subarbors, exhibiting variable branching and looping topology. Axonal branches did not overlap with each other within their respective parental subarbor nor with neurites of annexed subarbors. The sympathetic network in the border zone of a 2-week-old myocardial infarction was characterized by substantive rewiring, which included spatially heterogeneous loss and gain of sympathetic fibers and formation of multiple, predominately nested, axon loops of widely variable circumference and geometry. In contrast to mechanical tissue sectioning methods that may involve deformation of tissue and uncertainty in registration across sections, our approach preserves continuity of structure, which allows tracing of neurites over distances, and thus enables derivation of the three-dimensional and topological morphology of cardiac sympathetic nerves. Our assay should be of general utility to unravel the mechanisms governing sympathetic axon spacing during development and disease.
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