Vasculature in the mouse colon and spatial relationships with the enteric nervous system, glia, and immune cells

Abstract

The distribution, morphology, and innervation of vasculature in different mouse colonic segments and layers, as well as spatial relationships of the vasculature with the enteric plexuses, glia, and macrophages are far from being complete. The vessels in the adult mouse colon were stained by the cardiovascular perfusion of wheat germ agglutinin (WGA)-Alexa Fluor 448 and by CD31 immunoreactivity. Nerve fibers, enteric glia, and macrophages were immunostained in the WGA-perfused colon. The blood vessels entered from the mesentery to the submucosa and branched into the capillary networks in the mucosa and muscularis externa. The capillary net formed anastomosed rings at the orifices of mucosa crypts, and the capillary rings surrounded the crypts individually in the proximal colon and more than two crypts in the distal colon. Microvessels in the muscularis externa with myenteric plexus were less dense than in the mucosa and formed loops. In the circular smooth muscle layer, microvessels were distributed in the proximal, but not the distal colon. Capillaries did not enter the enteric ganglia. There were no significant differences in microvascular volume per tissue volume between the proximal and distal colon either in the mucosa or muscularis externa containing the myenteric plexus. PGP9.5-, tyrosine hydroxylase-, and calcitonin gene-related peptide (CGRP)-immunoreactive nerve fibers were distributed along the vessels in the submucosa. In the mucosa, PGP9.5-, CGRP-, and vasoactive intestinal peptide (VIP)-immunoreactive nerves terminated close to the capillary rings, while cells and processes labeled by S100B and glial fibrillary acidic protein were distributed mainly in the lamina propria and lower portion of the mucosa. Dense Iba1 immunoreactive macrophages were closely adjacent to the mucosal capillary rings. There were a few macrophages, but no glia in apposition to microvessels in the submucosa and muscularis externa. In conclusion, in the mouse colon, (1) the differences in vasculature between the proximal and distal colon were associated with the morphology, but not the microvascular amount per tissue volume in the mucosa and muscle layers; (2) the colonic mucosa contained significantly more microvessels than the muscularis externa; and (3) there were more CGRP and VIP nerve fibers found close to microvessels in the mucosa and submucosa than in the muscle layers.

Keywords: colon; immunohistochemistry; microvessel; mouse; vasculature; wheat germ agglutinin.

Figure 1

Vasculature of the mouse colon overviewed all layers from the serosa to the mucosa, labeled by WGA-AF 488 painting (cyan) and CD31 immunoreactivity (red) in an aboral segment of a mouse mid colon. Most of the microvessels were double-labeled (white). The vascular structures are similar in the mid and distal colon. (A) A 3D image of the flat wall of a mouse mid-colon with z-axis stacks. (B–D) Images are cropped from image (A) into three portions. (B) Capillary net in the mucosa near the lumen with the characteristic honeycomb-shaped rings connected together. (C) Vascular branches in the submucosal layer including the bottom of mucosal crypts. White arrows indicate the branches into the capillary nets at the bottom of mucosal crypts and magenta arrows indicate small veins. (D) Capillary net in the muscle layers near to the myenteric plexus. Blue arrows indicate the lymphatic vessels that had blind starting points (*), and wider and uneven diameters and valves (folds with brighter fluorescence). (E) A vertical section of the mid colon illustrates the location of layers cropped in images (B–D). Scale bars are at the bottom of each image (same as in all figures).

Figure 2

Vascular branches and connections in the mouse colon. The vessels were labeled by WGA painting (cyan in A, B, D and green in C) and CD31 immunoreactivity (red). (A) Submucosal vessels ramified and lead to capillary webs at the base of mucosal crypts. The cyan and white-colored vessels are the arterial branches till capillaries and red colored ones are veins. The sample was collected from the aboral segment of a mouse mid-colon. (B) Microphotograph shows that in the submucosa vessels from each side of the colon wall are connected, and the microvessels anastomosed in the networks near the antimesenteric margin (indicated by empty arrows) in a sample from the distal colon. (C) Microphotographs demonstrate that arterioles from the same artery lead into capillary networks in the mucosa (yellow arrows) and muscle layers (white arrows). The muscle layers in (C-1) and mucosa in (C-2) were cropped images from (C). Magenta arrows indicate the capillary networks at the bottom of mucosal crypts. (D) Microvascular networks were labeled complementary by WGA and CD31 in muscle layers. White arrows indicate a CD31-labeled microvessel that was not perfused well by WGA. Blue arrows mark a lymphatic vessel labeled by CD31. The samples for images (C, D) were sampled from a mouse proximal colon.

Figure 3

Mucosal microvessels in the mouse colon. (A, B) Transverse sections of a mouse proximal (Row A) and distal (Row B) colon. The vessels were painted by WGA perfusion (cyan) and CD31 immunoreactivity (red). The white arrows indicate the location of the capillary rings near the mucosal surface. Blue arrows: lymphatic vessels. (C–E) Magnification of microvessels in the proximal (C), mid (D), and distal colon (E). The framed areas in the right panels of rows (A, B) are magnified in (C, E), respectively. The capillaries connecting the capillary networks at the top and bottom of mucosal crypts were straighter in the mid and distal colon than in the proximal colon (A–E). (F–H) WGA (green)-painted capillary rings in the mucosa of the proximal colon (F), mid colon (G), and distal colon (H). Blue fluorescence is DAPI counterstaining. In the proximal colon, the capillary ring surrounded each mucosal crypt individually, while two crypts (see the DAPI-stained crypts) were in the distal colon. The proximal colon had more capillary rings, although smaller than the mid and distal colon in an area of the same size.

Figure 4

Microvessels in the muscle layers near the myenteric plexus in the mouse colon. Microvessels labeled by WGA perfusion (cyan) and CD31 immunoreactivity (red) in the muscle layers of the proximal (Row A) and distal colon (Row B). (C) The capillaries net (WGA, cyan) near the myenteric plexus (TH immunofluorescence, red) of the proximal colon. The capillaries passed through ganglion and interganglionic strands (marquee and arrows) and were magnified in (C'). The arrows indicate the capillary passing through. (D) Lymphatic vessels labeled with CD31 immunofluorescence with valves (blue arrows) in the muscle layer under the serosa. Several lymphatic vessels can also be seen in the panels of CD31 labeling in rows (A, B) (examples marked by blue arrows).

Figure 5

Quantifications of microvessels in the mouse colon. There were no significant differences between the proximal and distal colon when the microvessels were measured in 3D per tissue volume in three regions: mucosal microvessels in vertical sections (Mucosa), the top mucosal capillary rings (Capi top), and muscle layer near the myenteric plexus (Muscle). ^#p < 0.05 mucosa vs. muscle layers in the proximal and distal colon, respectively. N = 4–8 mice in each group as indicated in each bar.

Figure 6

Immunoreactive nerve fibers and vascular branches in the submucosa of mouse colon. The vessels were painted by WGA perfusion (cyan), and for the nerve-vessel relationship, WGA perfused samples were immunolabeled in red for PGP9.5, TH, CGRP, and VIP (red). The nerve fibers were not found in the arterioles entering capillary webs (examples are indicated by arrows). The pattern is similar in different colonic segments. The samples were collected from the distal colon.

Figure 7

A photomicrograph of triple fluorescent labeling of vessels and nerves in the submucosa of a mouse mid-colon shown in different panels with various fluorescent labeling. The vessels were painted by WGA perfusion (cyan) combined with CD31 immunolabeling (red), and nerves by PGP9.5 immunostaining (purple). The arteries (a), veins (v), and lymphatic vessels (lym) accompanied each other. WGA perfusion did not stain the veins. Nerve fibers traveled along the arteries. The lower-left panel is enlarged in the area framed in the panel earlier, demonstrating the nerve fibers traveling along the blood vessels. The single fine nerve fibers adjacent to the vascular wall (arrows) were possibly those innervating the vessels.

Figure 8

Nerve fibers and microvessels in the mucosa are shown in vertical sections of the mouse proximal colon. The vessels were painted by WGA perfusion (cyan) and PGP9.5, TH, CGRP, and VIP-ir nerve fibers were immunostained (red). More CGRP and VIP-ir than TH-ir nerve fibers are projected to the top of the mucosa (arrows). TH-ir terminals rarely reached the capillary rings at the mucosa top (arrows in the 2nd row). Yellow arrows in the top row indicate an arteriole in the lamina propria.

Figure 9

Examples illustrating nerve terminals distributed closely to the capillaries in the mucosa. Samples were from the proximal colon. Microphotographs at high magnification were acquired under 63X objective. (A–D) The vessels were painted by WGA perfusion (green), and nerve fibers were immunolabeled by PGP9.5, CGRP and VIP (red). (E, F) Show magnified areas framed in (D). The “***” indicated the lumen site.

Figure 10

Capillary rings and nerve fibers at the top of the mucosa. The samples were from the mid-colon. The vessels were painted by WGA perfusion (cyan), nerve fibers were immunolabeled by PGP9.5, CGRP, and VIP (red), and the tissues were counterstained by DAPI (blue). The different fluorescent channels merged in the same image demonstrate the mucosa capillary net in relation to immunofluorescent nerve terminals. High magnifications (objective 63X) of double labeling of WGA with each immunostaining are in the bottom panels.

Figure 11

Vessels and GFAP-ir glia in the mouse colon. The vessels were painted by WGA (cyan) and glial cells were immunostained by GFAP (red). Samples in (A–F) were from the mid-colon, and the sample in G–I was from the proximal colon. (A, B) GFAP-ir glia in the submucosal plexus (A) and myenteric plexus (B). The capillaries did not enter either of the plexuses. (C) GFAP-ir fibers surrounded vessels in the submucosa. (D–F) WGA-GFAP double labeling in the mucosa. (D) At the bottom of mucosal crypts; (E) capillary rings (WGA) near the lumen, no GFAP labeling found. (F) the red channel shows GFAP negative. (G–I) Portion of a mucosal fold from the proximal colon with WGA-painted microvessels and GFAP immunoreactive glial cells and processes. GFAP glia were not distributed around microvessels.

Figure 12

Vessels and S100B-ir cells in the mouse colon. The vessels were painted by WGA (cyan) and cells were immunostained by S100B (red). All the images are samples from the proximal colon. (A, B) S100B-ir cells in the submucosal plexus and myenteric plexus, respectively. The capillaries did not get into either of the plexuses. (C): S100B-ir cell bodies and processes surrounded vessels in the submucosa. (D) S100B-ir cells and microvessels in the circular muscle layer. (E) In the submucosal layer, S100B-ir cells formed in a net close to the capillary net at the bottom of mucosal crypts. (F) S100B-ir cells located close to the top mucosal capillary network. Arrow indicates S100B labels under the capillary rings. (G) Transverse section of a mucosal fold in the proximal colon showing S100B-ir cells located densely in the lower portion of mucosa and lamina propria (arrows). (H, I) High magnifications demonstrate some S100B-ir processes at the top of mucosal crypts while not in apposition to the capillaries. The image in (I) is tilted to show the locations of capillaries and S100B-ir processes (arrows).

Figure 13

Vessels and macrophages in the mouse colon. (A) Iba1-ir macrophages (red) distributed in the mucosa shown in a vertical section of the proximal colon (microvessels painted by WGA, cyan). (B) Dense Iba1-ir macrophages at the top of mucosa at high magnification. (C) WGA-pained vessels and Iba1-ir macrophages (red) in the capillary net at the bottom of mucosal crypts. (D) Iba1-ir macrophages close to the capillary net at the top of mucosal crypts. The three panels displayed the same photomicrography in different channel(s). (E) High magnification of Iba1-ir macrophages in the capillary rings at the bottom of the mucosal crypt. (F) High magnification of Iba1-ir macrophages in the capillary rings at the mucosal crypts top. (G) Iba1-ir macrophages in the submucosa. (H) Vessels (WGA+CD31) in a Peyer's patch with many Iba1-ir cells inside (purple). (I) Iba1-ir cells (purple) near the vessels (WGA cyan+CD31 red) in the submucosa. (J) Iba1-ir macrophages (cyan) in the muscularis externa with WGA and CD31 dual-labeled capillaries and a CD31-ir lymphatic vessel (red).