Investigation of heterocellular features of the mouse retinal neurovascular unit by 3D electron microscopy

Abstract

The retina has a complex structure with a diverse collection of component cells that work together to facilitate vision. The retinal capillaries supplying the nutritional requirements to the inner retina have an intricate system of neural, glial and vascular elements that interconnect to form the neurovascular unit (NVU). The retina has no autonomic nervous system and so relies on the NVU as an interdependent, physical and functional unit to alter blood flow appropriately to changes in the physiological environment. The importance of this is demonstrated by alterations in NVU function being apparent in the blinding disease diabetic retinopathy and other diseases of the retina. It is, therefore, imperative to understand the anatomy of the components of the NVU that underlie its functioning and in particular the nanoscale arrangements of its heterocellular components. However, information on this in three spatial dimensions is limited. In the present study, we utilised the technique of serial block-face scanning electron microscopy (SBF-SEM), and computational image reconstruction, to enable the first three-dimensional ultrastructural analysis of the NVU in mouse retinal capillaries. Mouse isolated retina was prepared for SBF-SEM and up to 150 serial scanning electron microscopy images (covering z-axes distances of 12-8 mm) of individual capillaries in the superficial plexus and NVU cellular components digitally aligned. Examination of the data in the x-, y- and z-planes was performed with the use of semi-automated computational image analysis tools including segmentation, 3D image reconstruction and quantitation of cell proximities. A prominent feature of the capillary arrangements in 3D was the extensive sheath-like coverage by singular pericytes. They appeared in close register to the basement membrane with which they interwove in a complex mesh-like appearance. Breaks in the basement membrane appeared to facilitate pericyte interactions with other NVU cell types. There were frequent, close (<10 nm) pericyte-endothelial interactions with direct contact points and peg-and-socket-like morphology. Macroglia typically intervened between neurons and capillary structures; however, regions were identified where neurons came into closer contact with the basement membrane. A software-generated analysis to assess the morphology of the different cellular components of the NVU, including quantifications of convexity, sphericity and cell-to-cell closeness, has enabled preliminary semi-quantitative characterisation of cell arrangements with neighbouring structures. This study presents new data on the nanoscale spatial characteristics of components of the murine retinal NVU in 3D that has implications for our understanding of structural integrity (e.g. pericyte-endothelial cell anchoring) and function (e.g. possible paracrine communication between macroglia and pericytes). It also serves as a platform to inform future studies examining changes in NVU characteristics with different biological and disease circumstances. All raw and processed image data have been deposited for public viewing.

Keywords: Neurovascular Unit; Peg-and-socket; SBF-SEM; astrocytes; basement membrane; capillaries; endothelium; macroglia; neurons; pericytes; retina.

FIGURE 1

Raw dataset for mouse retinal capillary. A collection of SBF‐SEM micrographs taken from a mouse retinal capillary to display the morphological changes along the capillary length. (a) Slice 1 (0.12 μm), (b) slice 50 (6 μm), (c) slice 100 (12 μm) and (d) slice 120 (14.4 μm), Lumen: L, Pericytes: P, Endothelium: E.

FIGURE 2

Segmented vasculature. The vasculature was identified, and each component was assigned a colour for segmentation through the data stack and subsequent visualisation in 3D reconstructions. (a) endothelium (aqua); (b) basement membrane (brown); (c) pericytes (blue); (d) complete vasculature.

FIGURE 3

3D reconstruction of mouse capillary vasculature. Displayed are features associated with a 3D reconstruction of the vasculature of capillary 1. Column a: endothelium (aqua); column b: basement membrane (brown); column c: pericytes (blue); and column d: combined vasculature.

FIGURE 4

Pericyte‐endothelial interaction via direct contact. Close contacts of a pericyte (blue) and endothelial cell (aqua) in absence of a bordering basement membrane (brown) are indicated for the raw data (a and c) and segmented data (b and d) of two sections 1.2 μm apart.

FIGURE 5

Peg‐and‐socket pericyte‐endothelial formation spanning across multiple sections. (a) Peg‐and‐socket formation identified on a capillary (magnified area evident on right hand side bounded by black box). (b–d) This peg and socket spanned across three sections (minimum 0.360 μm). (e) Displays the 3D nature of peg and sockets; corresponding video file can be found in File S15 (10.25405/data.ncl.19086197). Endothelium: aqua; basement membrane: brown; Pericytes: blue.

FIGURE 6

Distribution of peg and sockets across mouse capillaries. The distribution of peg‐and sockets evident across 150 sections examined in three capillaries. Note more than one peg‐and‐socket may appear in the same sections. (a) Two peg‐and‐sockets (indicated by white arrows) from mouse capillary 2 on slice 44 at depth 4.4 μm. (b) Additional peg from mouse capillary 2 on slice 61 at 6.1 μm depth. (c) Three peg‐and‐sockets from mouse capillary 2 on slice 89 at depth 8.9 μm. (d) A single peg‐and‐socket from mouse capillary 2 on slice 143 at depth 14.3 μm. (e) Peg‐and‐socket distribution graph for mouse capillaries 1, 2 and 3. Endothelium: aqua; basement membrane: brown; pericytes: blue. Scale bar: 3 μm.

FIGURE 7

Segmented data and 3D reconstruction of macroglia surrounding the vasculature. (a, b) Macroglia in (red and purple shades) can be seen to wrap around the vasculature across different sections along capillary depth. Purple shading depicts a cell identified as an astrocyte. Displayed in a and b are sections 6 μm apart. 3D reconstruction views (c, d) show the complexity of the wrapping along 18 of vessel (the corresponding video file can be found in File S17 (10.25405/data.ncl.19086341).

FIGURE 8

Macroglia‐pericyte direct contact. Top slide, raw data. Bottom slide, segmented features. Of the two macroglia evident in this image, represented in the bottom panel by purple and red shadings, one comes within close proximity of the pericyte (indicated in blue) by breaking through the BM (brown). An endothelial cell (aqua) is also shown.

FIGURE 9

Macroglia separation of the neurons from the vasculature. a, raw and b, segmented image showing neurons (green shades) typically separated from the vasculature by surrounding macroglia (red and purple) (Endothelium: aqua; basement membrane: brown; pericytes: blue). Such features can be viewed in the animated 3D reconstruction of File S22 (10.25405/data.ncl.19086350).

FIGURE 10

Neuron coming into close contact with vasculature. Raw (a and c) and segmented (b and d) data from consecutive sections (120 nm apart) illustrating a neuron in close proximity with the vasculature. (e and f) 3D arrangements from six consecutive sections: neurons: green shades, macroglia: red shades, endothelium: aqua; basement membrane: brown; pericytes: blue.

FIGURE 11

Quantitative assessment of intercellular and cellular‐BM proximities for capillary 1. (a) The stack mean population histogram of pericyte to endothelium distances. (b) The cumulative distribution function obtained from (a). (c) 10 nm readings of the cumulative distribution functions obtained from selected intercellular and cellular‐BM feature pairs to assess the degree of direct contact. BM, basement membrane; CDF, cumulative distribution function; E, endothelium; M, macroglia; N, neurons; P, pericyte.

FIGURE 12

Column scatter graphs for convexity, solidity and sphericity of NVU cellular components and the BM for capillary 1. All 150 slices were analysed with each dot representing the area‐weighted mean obtained for all segmented features within each slice. Panels (a–c) convexities (convex perimeter:perimeter, CP:P), solidities (area:convex area, A:CA) and sphericities (Rmin:Rmax) of NVU component cells and the BM. Further details of calculation can be found in “Methods” section.