Public Volume Electron Microscopy Data: An Essential Resource to Study the Brain Microvasculature

Abstract

Electron microscopy is the primary approach to study ultrastructural features of the cerebrovasculature. However, 2D snapshots of a vascular bed capture only a small fraction of its complexity. Recent efforts to synaptically map neuronal circuitry using volume electron microscopy have also sampled the brain microvasculature in 3D. Here, we perform a meta-analysis of 7 data sets spanning different species and brain regions, including two data sets from the MICrONS consortium that have made efforts to segment vasculature in addition to all parenchymal cell types in mouse visual cortex. Exploration of these data have revealed rich information for detailed investigation of the cerebrovasculature. Neurovascular unit cell types (including, but not limited to, endothelial cells, mural cells, perivascular fibroblasts, microglia, and astrocytes) could be discerned across broad microvascular zones. Image contrast was sufficient to identify subcellular details, including endothelial junctions, caveolae, peg-and-socket interactions, mitochondria, Golgi cisternae, microvilli and other cellular protrusions of potential significance to vascular signaling. Additionally, non-cellular structures including the basement membrane and perivascular spaces were visible and could be traced between arterio-venous zones along the vascular wall. These explorations revealed structural features that may be important for vascular functions, such as blood-brain barrier integrity, blood flow control, brain clearance, and bioenergetics. They also identified limitations where accuracy and consistency of segmentation could be further honed by future efforts. The purpose of this article is to introduce these valuable community resources within the framework of cerebrovascular research. We do so by providing an assessment of their vascular contents, identifying features of significance for further study, and discussing next step ideas for refining vascular segmentation and analysis.

Ultrastructure of Precapillary Sphincters and the Neurovascular Unit

“…An example of a PA branchpoint with mural cells encircling it has been shown previously in the MICrONS dataset(10) (Supp. Fig.…”

Pericyte heterogeneity identified by 3D ultrastructural analysis of the microvessel wall

“…Other plausible cell types include microglia (Bisht et al, 2021) and perhaps astrocytes, but these cells typically associate with the cerebrovasculature via cellular extensions or processes (e.g., astrocytic end-feet) without outright incorporation in the vessel wall and within the vBM/ basal lamina. Perivascular microglia extend thin cellular processes in numerous directions (Bonney et al, 2022a), with very few, if any, actually ensheathing the vessel wall or incorporating within the continuous band of ECM that surrounds the vessel circumference. Additionally, classification of these cells as "perivascular fibroblasts" (PVFs) seems unlikely based on observations demonstrating that mouse brain PVFs are highly dynamic in their movement along the vessel wall (Bonney et al, 2022b), which does not align well with a cell that appears to be embedded in the ECM surrounding the vessel wall.…”

“…It remains to be determined however if PCs and ASCs form cell-cell contacts/ junctions, or if they are separated by the vBM at all locations and depend solely on paracrine signaling for their communication. Cells involved in immune surveillance have also been proposed to reside adjacent to blood vessels including microglia (Bisht et al, 2021), perivascular macrophages (Faraco et al, 2016;Faraco et al, 2017), and perivascular fibroblasts (Ornelas et al, 2021;Bonney et al, 2022a;Bonney et al, 2022b), but the extent to which these cells may be located within the vBM, if at all, is still being established.…”

Estimates for the astrocyte endfoot sheath permeability of the extra-cellular pathway

“…2 µm for a penetrating vessel with r v = 10 µm. Finally, we added the thickness of the endothelial cell layer and basement membrane with 0.4 µm [35] for all vessels. In summary, r o = 2 h ES ( r v )+0.4 = 1.3 r v +1.5 for r v ≥ 3 µm and r o = h ES ( r v )+0.4 = 1.15 r v +0.95 otherwise.…”