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Different segments of the cerebral vasculature reveal specific endothelial specifications, while tight junction proteins appear equally distributed

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Abstract

The identification of the “paucity of transportation vesicles” and “belt-like” tight junctions (TJs) of endothelial cells as the “morphological correlate of a blood–brain barrier” (BBB) by Reese and Karnovsky (J Cell Biol 34:207–217, 1967) has become textbook knowledge, and countless studies have helped to further define the elements, functions, and dynamics of the BBB. Most work, however, has focused on parenchymal capillaries or less clearly defined “microvessels”, while a systematic study on similarities and differences between BBB architecture along the vascular tree within the brain and the meninges has been lacking. Since astrocytes induce endothelial cells to display BBB-typical characteristics by sonic hedgehog and Wnt/β-catenin signaling, we hypothesized that BBB-typical features should be most pronounced in parenchymal capillaries, where endothelium and astrocytes are separated by a basement membrane only. In contrast, this intimate contact is absent in leptomeningeal vessels, thereby potentially affecting BBB architecture. However, here, we show that claudin-3, claudin-5, zonula occludens-1, and occludin as typical constitutes of BBB TJs are comparably distributed in all segments of the parenchymal and the meningeal vascular tree of C57Bl6 mice. While electron microscopy revealed equally occluded interendothelial clefts, arterial vessels of the brain parenchyma but not within the meninges exhibited significantly longer TJ overlaps compared to capillaries. The highest density of endothelial vesicles was found in arterial vessels. Thus, endothelial expression of BBB-typical TJ proteins is not reflected by the distance to surrounding astrocytes, but electron microscopy reveals significant differences of endothelial specification along different segments of the CNS vasculature.

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Acknowledgments

The authors would like to thank Judith Craatz and Jana Brendler (Institute of Anatomy, University of Leipzig) for technical assistance in tissue preparation. Funding: Deutsche Forschungsgemeinschaft for funding (DFG-FOR 1336), Helmholtz Alliance ICEMED (Imaging and Curing Environmental Metabolic Diseases).

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    1. Institute of Anatomy, University of Leipzig, Liebigstraße 13, 04103, Leipzig, Germany

      Sophie Hanske, Felix Dyrna, Ingo Bechmann & Martin Krueger

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    1. Sophie Hanske
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    Correspondence to Ingo Bechmann or Martin Krueger.

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    Deutsche Forschungsgemeinschaft for funding (DFG-FOR 1336), Helmholtz Alliance ICEMED (Imaging and Curing Environmental Metabolic Diseases).

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    S. Hanske and F. Dyrna contributed equally.

    I. Bechmann and M. Krueger contributed equally.

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    429_2016_1267_MOESM1_ESM.tif

    Supplementary Fig. 1 Expression pattern of occludin and claudin-5 was further assessed in parenchymal arteries, capillaries, and veins. Therefore, the length of individual occludin-positive TJ strands was measured and compared with the length positive for claudin-5 within the same TJ strand. Thus, a ratio of the occludin-positive TJ strand length covered by immunoreactivity for claudin-5 was used to check for differences in the expression pattern between arteries (A), capillaries (C), and veins (V). Here, differences impressively failed to reach statistical significance (p = 0.18, Kruskal–Wallis test; Dunn’s multiple comparison; n = 5; bars are given as standard deviation of the mean). Capillaries, veins, and arteries were differentiated by diameter and immunoreactivity for α-SMA. Scale bar: 10 µm; DAPI: nuclei (TIFF 4133 kb)

    429_2016_1267_MOESM2_ESM.tif

    Supplementary Fig. 2 Electron micrograph illustrating the topography of perivascular spaces, here surrounding an arterial vessel. Higher magnification (right) allows clear-cut delineation of the different cellular populations and basal laminas. Of note, PVS are bordered by the outermost vascular basal lamina (highlighted in yellow) and the glial (astrocytic) basal lamina (highlighted in red). Pericytes (not shown) or smooth muscle cells (SMC) are by definition part of the vascular wall, ensheathed by the vascular basal laminas, whereas leptomeningeal mesothelial cells (arrow heads) or perivascular macrophages (not shown) are located within PVS. ‘Spaces’ around parenchymal vessels, which are not bordered by basal laminas that are often related to perfusion- or dehydration-related swelling of astrocytic endfeet, thus representing artifacts. E = endothelium, L = vascular lumen, arrow = endothelial TJ (TIFF 2305 kb)

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    Hanske, S., Dyrna, F., Bechmann, I. et al. Different segments of the cerebral vasculature reveal specific endothelial specifications, while tight junction proteins appear equally distributed. Brain Struct Funct 222, 1179–1192 (2017). https://doi.org/10.1007/s00429-016-1267-0

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