Investigation of heterocellular features of the mouse retinal neurovascular unit by 3D electron microscopy.
Investigation of heterocellular features of the mouse retinal neurovascular unit by 3D electron microscopy.
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通过3D电子显微镜研究小鼠视网膜神经血管单元的杂细胞特征。
作者：
视网膜具有复杂的结构，具有多种组成细胞，这些细胞共同作用以促进视力。为视网膜内层提供营养需求的视网膜毛细血管具有复杂的神经、神经胶质和血管元件系统，这些元件相互连接形成神经血管单元（NVU）。视网膜没有自主神经系统，因此依赖 NVU 作为一个相互依赖的物理和功能单位来适当地改变血流以适应生理环境的变化。 NVU 功能的改变在致盲疾病糖尿病性视网膜病和其他视网膜疾病中表现得很明显，这证明了这一点的重要性。因此，必须了解构成其功能的 NVU 组件的解剖结构，特别是其异质细胞组件的纳米级排列。然而，关于这三个空间维度的信息是有限的。在本研究中，我们利用串行块面扫描电子显微镜 (SBF-SEM) 和计算图像重建技术，首次对小鼠视网膜毛细血管中的 NVU 进行三维超微结构分析。小鼠离体视网膜准备用于 SBF-SEM 和多达 150 个浅神经丛中单个毛细血管和数字对齐的 NVU 细胞成分的串行扫描电子显微镜图像（覆盖 z 轴距离 12-8mm）。使用半自动计算图像分析工具（包括分割、3D 图像重建和细胞邻近度定量）对 x、y 和 z 平面中的数据进行检查。 3D 毛细血管排列的一个显着特征是单个周细胞的广泛鞘状覆盖。它们看起来与基底膜紧密对齐，并与基底膜交织成复杂的网状外观。基底膜的破裂似乎促进了周细胞与其他 NVU 细胞类型的相互作用。周细胞与内皮之间存在频繁、紧密（<10 nm）的相互作用，具有直接接触点和钉窝状形态。大胶质细胞通常介入神经元和毛细血管结构之间；然而，我们发现了神经元与基底膜接触更紧密的区域。软件生成的分析用于评估 NVU 不同细胞成分的形态，包括凸度、球形度和细胞间紧密度的量化，使得能够对邻近结构的细胞排列进行初步半定量表征。这项研究提供了关于 3D 小鼠视网膜 NVU 组件的纳米级空间特征的新数据，这对我们理解结构完整性（例如周细胞-内皮细胞锚定）和功能（例如大胶质细胞和周细胞之间可能的旁分泌通讯）具有重要意义。它还可以作为一个平台，为未来研究不同生物和疾病环境下 NVU 特征的变化提供信息。所有原始和处理后的图像数据均已保存以供公众查看。在本研究中，我们提供了小鼠视网膜神经血管单元（NVU）的三维超微结构分析。人们发现周细胞以鞘状方式与基底膜交织，同时与毛细血管内皮细胞进行定期的钉和插座状相互作用。观察到同细胞和异细胞（带有神经元）的回旋大胶质细胞沿着毛细血管长度交织。有时，大胶质细胞和神经元细胞分别靠近周细胞和基底膜。本文揭示的 NVU 复杂的纳米级空间特征将为未来研究不同生物和疾病环境下 NVU 结构和功能的变化提供信息。
The retina has a complex structure with a diverse collection of component cells that work together to facilitate vision. The retinal capillaries supplying the nutritional requirements to the inner retina have an intricate system of neural, glial and vascular elements that interconnect to form the neurovascular unit (NVU). The retina has no autonomic nervous system and so relies on the NVU as an interdependent, physical and functional unit to alter blood flow appropriately to changes in the physiological environment. The importance of this is demonstrated by alterations in NVU function being apparent in the blinding disease diabetic retinopathy and other diseases of the retina. It is, therefore, imperative to understand the anatomy of the components of the NVU that underlie its functioning and in particular the nanoscale arrangements of its heterocellular components. However, information on this in three spatial dimensions is limited. In the present study, we utilised the technique of serial block‐face scanning electron microscopy (SBF‐SEM), and computational image reconstruction, to enable the first three‐dimensional ultrastructural analysis of the NVU in mouse retinal capillaries. Mouse isolated retina was prepared for SBF‐SEM and up to 150 serial scanning electron microscopy images (covering z‐axes distances of 12–8 mm) of individual capillaries in the superficial plexus and NVU cellular components digitally aligned. Examination of the data in the x‐, y‐ and z‐planes was performed with the use of semi‐automated computational image analysis tools including segmentation, 3D image reconstruction and quantitation of cell proximities. A prominent feature of the capillary arrangements in 3D was the extensive sheath‐like coverage by singular pericytes. They appeared in close register to the basement membrane with which they interwove in a complex mesh‐like appearance. Breaks in the basement membrane appeared to facilitate pericyte interactions with other NVU cell types. There were frequent, close (<10 nm) pericyte–endothelial interactions with direct contact points and peg‐and‐socket‐like morphology. Macroglia typically intervened between neurons and capillary structures; however, regions were identified where neurons came into closer contact with the basement membrane. A software‐generated analysis to assess the morphology of the different cellular components of the NVU, including quantifications of convexity, sphericity and cell‐to‐cell closeness, has enabled preliminary semi‐quantitative characterisation of cell arrangements with neighbouring structures. This study presents new data on the nanoscale spatial characteristics of components of the murine retinal NVU in 3D that has implications for our understanding of structural integrity (e.g. pericyte‐endothelial cell anchoring) and function (e.g. possible paracrine communication between macroglia and pericytes). It also serves as a platform to inform future studies examining changes in NVU characteristics with different biological and disease circumstances. All raw and processed image data have been deposited for public viewing. In the present study, we provide a three‐dimensional ultrastructural analysis of the mouse retinal neurovascular unit (NVU). Pericytes were found to interweave in a sheath‐like fashion with the basement membrane while making regular peg‐and‐socket‐like interactions with capillary endothelial cells. Convoluted macroglial homo‐ and heterocellular (with neurones) interweaving along the capillary length was observed. On occasions macroglial and neuronal cells, respectively, came in close proximity to pericytes and the basement membrane. The complex nanoscale spatial characteristics of the NVU revealed herein will inform future studies examining changes in NVU structure and function with different biological and disease circumstances.
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影响因子：
3.5
作者：
Agrawal SA;Burgoyne T;Eblimit A;Bellingham J;Parfitt DA;Lane A;Nichols R;Asomugha C;Hayes MJ;Munro PM;Xu M;Wang K;Futter CE;Li Y;Chen R;Cheetham ME
通讯作者：
Cheetham ME
影响因子：
5.3
作者：
Kerov, Vasily;Laird, Joseph G.;Lee, Amy
通讯作者：
Lee, Amy
DOI：
10.3129/i08-056
发表时间：
2008-06-01
影响因子：
4.2
作者：
Flammer, Josef;Mozaffarieh, Maneli
通讯作者：
Mozaffarieh, Maneli
影响因子：
2.4
作者：
Ochs, M;Mayhew, TM;Knabe, W
通讯作者：
Knabe, W
影响因子：
9.8
作者：
Belevich I;Joensuu M;Kumar D;Vihinen H;Jokitalo E
通讯作者：
Jokitalo E