Organizational hierarchy and structural diversity of microvascular pericytes in adult mouse cortex

Abstract

Smooth muscle cells and pericytes, together called mural cells, coordinate many distinct vascular functions. Canonically, smooth muscle cells are ring-shaped and cover arterioles with circumferential processes, whereas pericytes extend thin processes that run longitudinally along capillaries. In between these canonical mural cell types are cells with features of both smooth muscle cells and pericytes. Recent studies suggest that these transitional cells are critical for controlling blood flow to the capillary bed during health and disease, but there remains confusion on how to identify them and where they are located in the brain microvasculature. To address this issue, we measured the morphology, vascular territory, and α-smooth muscle actin content of structurally diverse mural cells in adult mouse cortex. We first imaged intact 3D vascular networks to establish the locations of major gradations in mural cell appearance as arterioles branched into capillaries. We then imaged individual mural cells occupying the regions within these gradations. This revealed two transitional cells that were often similar in appearance, but with sharply contrasting levels of α-smooth muscle actin. Our findings highlight the diversity of mural cell morphologies in brain microvasculature, and provide guidance for identification and categorization of mural cell types.

Keywords: Blood flow; capillary; microvasculature; mural cell; pericyte.

Figure 1.

Mural cell organization and vascular structure revealed in optically cleared mouse cortex. (a) Reconstructed volume from barrel cortex of a PDGFRβ-tdTomato mouse, showing tdTomato fluorescence. (b) The same tissues were immunolabeled with FITC-conjugated α-SMA antibody. (c) Composite image of tdTomato and FITC channels. (d) Schematic showing the terms used to describe various portions of the vascular anatomy (left side), and the system for ordering microvessel branches as they ramified from the penetrating arteriole (right side).

Figure 2.

Minimal variation of pericyte features with penetrating arteriole offshoots at different cortical depths. Penetrating arterioles imaged in PDGFRβ-tdTomato mouse cortex. (a) The first ovoid cell body on a penetrating arteriole offshoot (arrowhead). The numbers denote branch order, as defined in Figure 1(d). (b) Examples of α-SMA termini, where α-SMA labeling decreases sharply (arrows). (c) Example of a coverage shift (arrowhead), where thin, longitudinal running processes are first observed. (d–f) Histograms showing the frequency at which each mural cell feature occurs at each branch order. (g–i) Scatterplots of each mural cell feature, showing branch order of occurrence as a function of cortical depth. Running average (window size 200 µm; step size 50 µm) ± SEM is shown.

Figure 3.

α-SMA content and pericyte coverage extend further along larger penetrating arteriole offshoots. Penetrating arterioles imaged in PDGFRβ-tdTomato mouse cortex. First order branches of penetrating arteriole offshoots range in diameter. Examples of small (a) and large (b) branches, with diameters of 8 µm and 16 µm at their points of emergence, respectively. Note that α-SMA does not extend into the small branch, while the large branch supports α-SMA for several branch orders. (c–e) Scatterplots of each mural cell feature, showing average branch order of occurrence as a function of the 1st order branch diameter. Analysis was performed with Pearson’s correlation; n = 52 penetrating arteriole offshoots, collected over seven penetrating arterioles from two mice.

Figure 4.

α-SMA content of mural cell types in sparsely labeled NG2-tdTomato mice. (a) Wide field view of penetrating arteriole in barrel cortex of NG2-tdTomato mouse. Mural cells are labeled with tdTomato (red) and vascular endothelium was labeled with FITC-conjugated lectin (green). Images were captured from 100 to 200 µm thick coronal brain sections using confocal microscopy. (b) The same region of tissue showing immunolabel with α-SMA antibody. (c) A smooth muscle cell (SMC) observed on the 0th order penetrating arteriole. An α-SMA antibody and FITC-lectin co-label is also shown. (d) A representative ensheathing pericyte (EP) on a pre-capillary arteriole. White arrows point to ovoid cell bodies. (e) A representative mesh pericyte (MP) on a capillary. Note that the cell abuts the α-SMA terminus. (f) A typical thin-strand pericyte (TSP), the canonical form of pericyte, on a capillary. (g) Intensity of α-SMA exhibited for each mural cell groups. ***p < 0.001, Kruskal–Wallis H test with Dunn-Bonferroni post hoc test; n = 15 each for SMC, EP, MP, and TSP. Data were collected over three mice. Data shown as mean ± SEM. Images of α-SMA staining are raw, with no correction for background fluorescence.

Figure 5.

Mural cell types exhibit varying cell lengths and degrees of vessel coverage. (a–d) Examples of each mural cell type from NG2-tdTomato mice, with lines to measure total cell length (gray lines) and vessel diameter (white lines). (e–h) Vessel coverage for each mural cell was calculated by dividing tdTomato-positive area (red region) by a mask of the vessel area underlying the cell. The black regions show portions of the mask that are not covered by the mural cell. (i,j) Total cell length and vessel coverage for each mural cell group. *p < 0.05, **p < 0.01, ***p < 0.001, Kruskal–Wallis H test with Dunn-Bonferroni post hoc test; n = 20 each for SMC, EP, MP, and TSP. Data were collected over three mice. Data shown as mean ± SEM.

Figure 6.

Organizational hierarchy for mural cell types in the mouse cortical vasculature and flow chart for classification. (a) Schematic showing transition of mural cell types as penetrating arteriole offshoots transition from pre-capillary arterioles to capillaries. The names of mural cell groups, features examined in this study, and approximate branch orders of occurrence are depicted. “Mesh pericytes” and “thin-strand pericytes” describe the appearance of cells within the broader category of “capillary pericytes,” though they could not be statistically separated using the metrics collected in this study. (b) Flowchart for distinguishing three major mural cells groups, smooth muscle cells, ensheathing pericytes, and capillary pericytes, the latter of which consists of mesh and thin-strand pericytes.