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. 2024 Aug 26:2024:4482931.
doi: 10.1155/2024/4482931. eCollection 2024.

The Blood-Brain Barrier in Both Humans and Rats: A Perspective From 3D Imaging

Aiwen Chen  1   2   3   4   5 , Gavin Volpato  6   7 , Alice Pong  6   7 , Emma Schofield  6   7 , Jun Huang  8 , Zizhao Qiu  9 , George Paxinos  6   7 , Huazheng Liang  3   6   9
Affiliations

The Blood-Brain Barrier in Both Humans and Rats: A Perspective From 3D Imaging

Aiwen Chen et al. Int J Biomed Imaging. .

Abstract

Background: The blood-brain barrier (BBB) is part of the neurovascular unit (NVU) which plays a key role in maintaining homeostasis. However, its 3D structure is hardly known. The present study is aimed at imaging the BBB using tissue clearing and 3D imaging techniques in both human brain tissue and rat brain tissue. Methods: Both human and rat brain tissue were cleared using the CUBIC technique and imaged with either a confocal or two-photon microscope. Image stacks were reconstructed using Imaris. Results: Double staining with various antibodies targeting endothelial cells, basal membrane, pericytes of blood vessels, microglial cells, and the spatial relationship between astrocytes and blood vessels showed that endothelial cells do not evenly express CD31 and Glut1 transporter in the human brain. Astrocytes covered only a small portion of the vessels as shown by the overlap between GFAP-positive astrocytes and Collagen IV/CD31-positive endothelial cells as well as between GFAP-positive astrocytes and CD146-positive pericytes, leaving a big gap between their end feet. A similar structure was observed in the rat brain. Conclusions: The present study demonstrated the 3D structure of both the human and rat BBB, which is discrepant from the 2D one. Tissue clearing and 3D imaging are promising techniques to answer more questions about the real structure of biological specimens.

Keywords: 3D imaging; astrocyte; basal membrane; blood-brain barrier; endothelium; microglia; tissue clearing.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
(a, b) Immunofluorescence staining against Collagen IV to show blood vessels in the human brain. Note the area with little collagen-positive signal (arrows). The scale bar is 200 μm in (a) and 100 μm in (b).
Figure 2
Figure 2
(a, b) Immunofluorescence staining against Glut1 to show blood vessels in the human brain. Note the patches with little Glut1-positive signal (arrows). The scale bar is 100 μm in (a) and 50 μm in (b).
Figure 3
Figure 3
(a, b) Immunofluorescence staining against Iba1 to show microglial cells in the human brain. Note the thick branches (arrows) and thin processes (arrowheads). The scale bar is 200 μm in (a) and 50 μm in (b).
Figure 4
Figure 4
(a, b) Immunofluorescence staining against CD31 (endothelial cells) and GFAP (astrocytes) to show their spatial relationship in the human brain. Note the processes (arrowheads) of astrocytes wrapping blood vessels (arrows) and their overlap (arrowheads). The scale bar is 200 μm in (a) and 50 μm in (b).
Figure 5
Figure 5
(a, b) Immunofluorescence staining against CD31 (endothelial cells) and GFAP (astrocytes) to show their spatial relationship in the rat brain. Note the processes (arrowheads) of astrocytes wrapping blood vessels and their overlap. Patches of blood vessels without CD31-positive signal were observed (arrows). The scale bar is 20 μm in (a) and 10 μm in (b).
Figure 6
Figure 6
(a, b) Immunofluorescence staining against CD146 (pericytes) and GFAP (astrocytes) to show their spatial relationship in the rat hippocampus. Note the overlap between processes of astrocytes and pericytes (arrows). The scale bar is 150 μm in (a) and 50 μm in (b).
See this image and copyright information in PMC

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