High-resolution electron microscopy (HREM) imaging of the in vitro blood-brain barrier (BBB), is a promising modality for investigating the dynamic morphological interplay underpinning BBB development. The successful establishment of BBB integrity is grounded in the brain endothelial cells (BEC’s) ability to occlude its paracellular spaces of brain capillaries through the expression of the intercellular tight junction (TJ) proteins. The impermeability of these paracellular spaces are crucial in the regulation of transcellular transport systems to achieve homeostasis of the central nervous system. To-date research describing morphologically, the dynamics by which TJ interaction is orchestrated to successfully construct a specialized barrier remains undescribed. In this study, the application of HREM illuminates the novel, dynamic and highly restrictive BEC paracellular pathway which is founded based on lateral membrane alignment which is the functional imperative for the mechanical juxtapositioning of TJ zones that underpin molecular bonding and sealing of the paracellular space. For the first time, we report on the secretion of a basement membrane in vitro, which allow BECs to orientate themselves into distinct basolateral and apicolateral domains and establish a 3-dimensional BEC construct. We report for the first time, on the expression of nanovesicles bound to the plasma membrane surfaces of the BECs. These membrane-bound vesicles are reported to possess an array of DNA/RNA constituents and chemotaxic properties affecting the formation of nanotubes that span the paracellular space between BECs, facilitating BBB construction, alluding to a functional role in mediating cell-to-cell communication. This study suggests that novel, ultrathin nanotubular (NT) structures are involved in functional roles in bringing into alignment the paracellular space of BECs. Immortalized mouse BECs (b.End3, b.End5) and primary rat cardiac microvascular ECs were used to further validate the in vitro BBB model by profiling variances in peripheral EC monolayer development. These cells presented with an opposite topographical profile: large fenestra and intercellular spaces, devoid of morphological ultrastructures. This comparative study alludes to the role of NT facilitation in TJ-induced hemifusion of apicolateral BEC membranes, as a structural event forming the basis for establishing a polarized BBB.

中文翻译：

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对体外发展的血脑屏障的高分辨率见解：内皮纳米管功能的新形态特征

体外血脑屏障（BBB）的高分辨率电子显微镜（HREM）成像是一种有前途的方式，用于研究支撑血脑屏障发展的动态形态相互作用。BBB完整性的成功建立基于大脑内皮细胞（BEC's）通过表达细胞间紧密连接（TJ）蛋白来阻塞其大脑毛细血管旁细胞空间的能力。这些旁细胞间隙的不可渗透性在调节跨细胞转运系统以实现中枢神经系统的稳态方面是至关重要的。迄今为止，从形态学上描述的研究还没有描述通过TJ相互作用成功构建特化屏障的动力学。在这项研究中，HREM的应用阐明了该小说，动态和高度限制性的BEC旁细胞通路是基于侧向膜排列建立的，这是对TJ区域进行机械并置的必要功能，TJ区域是分子间结合和密封的基础。首次，我们报道了体外基底膜的分泌，该分泌使BEC能够将自身定向为独特的基底外侧和apicolateral区域，并建立3维BEC构建体。我们首次报道了与BECs质膜表面结合的纳米囊泡的表达。据报道，这些与膜结合的囊泡具有一系列DNA / RNA成分，其化学趋化特性会影响跨越BEC之间的细胞旁空间的纳米管的形成，从而促进BBB的构建，暗示在介导细胞间通讯中的功能性作用。这项研究表明，新颖的超薄纳米管（NT）结构参与使BEC的细胞旁空间对齐的功能性作用。使用永生化的小鼠BEC（b.End3，b.End5）和原代大鼠心脏微血管EC通过分析外周EC单层发育的差异来进一步验证体外BBB模型。这些细胞具有相反的形貌特征：大的窗孔和细胞间空间，没有形态的超微结构。这项比较研究暗示了NT促进作用在TJ诱导的apicolateral BEC膜半融合中的作用，这是构成建立极化BBB基础的结构事件。超薄纳米管（NT）结构参与使BEC的细胞旁空间排列一致的功能。使用永生化的小鼠BEC（b.End3，b.End5）和原代大鼠心脏微血管EC通过分析外周EC单层发育的差异来进一步验证体外BBB模型。这些细胞具有相反的形貌特征：大的窗孔和细胞间空间，没有形态的超微结构。这项比较研究暗示了NT促进作用在TJ诱导的apicolateral BEC膜半融合中的作用，这是构成建立极化BBB基础的结构事件。超薄纳米管（NT）结构参与使BEC的细胞旁空间排列一致的功能。使用永生化的小鼠BEC（b.End3，b.End5）和原代大鼠心脏微血管EC通过分析外周EC单层发育的差异来进一步验证体外BBB模型。这些细胞具有相反的形貌特征：大的窗孔和细胞间空间，没有形态的超微结构。这项比较研究暗示了NT促进作用在TJ诱导的apicolateral BEC膜半融合中的作用，这是构成建立极化BBB基础的结构事件。End5）和原代大鼠心脏微血管EC用于通过分析外周EC单层发育的差异进一步验证体外BBB模型。这些细胞具有相反的形貌特征：大的窗孔和细胞间空间，没有形态的超微结构。这项比较研究暗示了NT促进作用在TJ诱导的apicolateral BEC膜半融合中的作用，这是构成建立极化BBB基础的结构事件。End5）和原代大鼠心脏微血管EC用于通过分析外周EC单层发育的差异进一步验证体外BBB模型。这些细胞具有相反的形貌特征：大的窗孔和细胞间空间，没有形态的超微结构。这项比较研究暗示了NT促进作用在TJ诱导的apicolateral BEC膜半融合中的作用，这是构成建立极化BBB基础的结构事件。