インビボイメージング法を用いたマウス小腸の肉芽組織深部における腸壁内神経形成の解析

One of the challenges of using imaging techniques as a tool to study cellular physiology has been the inability to resolve structures that are not located near the surface of the preparation. Nonlinear optical microscopy, in particular two photon-excited fluorescence microscopy (2PM), has overcome this limitation, providing deeper optical penetration (several hundred µm) in ex vivo and in vivo preparations. We have used this approach in the gut to achieve the first in vivo imaging of enteric neurons and nerve fibers in the mucosa, submucosa, submucosal and myenteric plexuses, and circular and longitudinal muscles of the small intestine in H-line: Thy1 promoter GFP mice. Moreover, we obtained clear three-dimensional imaging of enteric neurons that were newly generated after gut transection and reanastomosis. Neurogenesis was promoted by oral application of the 5-HT4-receptor agonist, mosapride citrate (MOS). The number of newly generated neurons observed in mice treated with MOS for one week was 421±89 per 864,900 µm2 (n = 5), which was significantly greater than that observed in preparations treated with MOS plus an antagonist (113±76 per 864,900 µm2) or in 4 week vehicle controls (100±34 per 864,900 µm2) (n = 4 both). Most neurons were located within 100 µm of the surface. These results confirm that activation of enteric neural 5-HT4-receptor by MOS promotes formation of new enteric neurons. We conclude that in vivo 2PM imaging made it possible to perform high-resolution deep imaging of the living mouse whole gut and reveal formation of new enteric neurons promoted by 5-HT4-receptor activation.博士（医学）・甲第631号・平成27年3月16日© 2014 Goto Kei et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

In vivo imaging of enteric neurogenesis in the deep tissue of mouse small intestine.

One of the challenges of using imaging techniques as a tool to study cellular physiology has been the inability to resolve structures that are not located near the surface of the preparation. Nonlinear optical microscopy, in particular two photon-excited fluorescence microscopy (2PM), has overcome this limitation, providing deeper optical penetration (several hundred µm) in ex vivo and in vivo preparations. We have used this approach in the gut to achieve the first in vivo imaging of enteric neurons and nerve fibers in the mucosa, submucosa, submucosal and myenteric plexuses, and circular and longitudinal muscles of the small intestine in H-line: Thy1 promoter GFP mice. Moreover, we obtained clear three-dimensional imaging of enteric neurons that were newly generated after gut transection and reanastomosis. Neurogenesis was promoted by oral application of the 5-HT(4)-receptor agonist, mosapride citrate (MOS). The number of newly generated neurons observed in mice treated with MOS for one week was 421±89 per 864,900 µm(2) (n = 5), which was significantly greater than that observed in preparations treated with MOS plus an antagonist (113±76 per 864,900 µm(2)) or in 4 week vehicle controls (100±34 per 864,900 µm(2)) (n = 4 both). Most neurons were located within 100 µm of the surface. These results confirm that activation of enteric neural 5-HT(4)-receptor by MOS promotes formation of new enteric neurons. We conclude that in vivo 2PM imaging made it possible to perform high-resolution deep imaging of the living mouse whole gut and reveal formation of new enteric neurons promoted by 5-HT(4)-receptor activation

Images of anastomotic region of the terminal ileum in a MOS-treated mouse.

<p>The dotted lines indicates the anastomosis site. Around the knot of thread we obtained each image from 9 visual fields. <b><i>A.</i></b> Images stacked with Z axis to a total depth of 200–300 µm. <b><i>A–a.</i></b> image 42 µm deep to the serosa surface in area (<b><i>a</i></b>) in <b><i>A</i></b>. <b><i>A–a’.</i></b> image 174 µm deep to the serosa surface in the same area (<b><i>a</i></b>) in <b><i>A</i></b>. <b><i>A–b.</i></b> 44 µm deep to the serosa surface in area (<b><i>b</i></b>) in <b><i>A</i></b>. <b><i>A–b’.</i></b> image 101 µm deep to the serosa surface in the same area (<b><i>b</i></b>) in <b><i>A</i></b>. Arrows indicate nerve cells in <b><i>A–a’</i></b>, <b><i>b</i></b> and <b><i>b’</i></b>, and arrowheads indicate nerve fibers in <b><i>A–a</i></b>, <b><i>a’</i></b>, <b><i>b</i></b> and <b><i>b’</i></b>, and circles indicate ganglion-like clusters of neurons in <b><i>A–a</i></b>, <b><i>b</i></b> and <b><i>b’</i></b>, respectively. <b><i>B.</i></b> Number of neurons in each field (size: 310 µm×310 µm) around the knot. <b><i>C.</i></b> Newborn nerve cells formed ganglion structures indicated by circles. These were enlarged from the images shown in <b><i>A–b’–i</i></b> and <b><i>–ii</i></b>.</p

Images of anastomosis of the ileum in an SB-207266 (SB) plus MOS treated mouse.

<p>SB plus MOS treatment was performed for one week. <b><i>A.</i></b> Images stacked in the Z axis with a total depth of 200 - 300 µm. <b><i>A–a.</i></b> image 38 µm deep to the serosa surface in area (<b><i>a</i></b>) in <b><i>A</i></b>. <b><i>A–b</i></b>. image 71 µm deep to the serosa surface in area (<b><i>b</i></b>) in <b><i>A</i></b>. Circles indicate aggregates of small non-neuronal cells (<b><i>A–a</i></b> and <b><i>b</i></b>), respectively.</p

Immunohistochemical image for anti-neurofilament (NF) antibody of a whole mount preparation of the same intestine shown in <b>Figure 5</b>.

<p><i>A–a</i> corresponds to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054814#pone-0054814-g005" target="_blank">Figure 5<i>A</i></a> (the image by 2PM). *, A knot of thread in the area between two-dotted lines indicates the anastomotic area. The granulation tissue was removed to allow for laser penetration. Normal myenteric plexus in the intact oral and anal sites are visible, but nerve cells and fibers are not visible in the anastomotic region because of the thickness of the anastomotic area.</p

Images of around the suture knot at anastomosis of the ileum in DMSO treated mouse.

<p>Vehicle treatments were performed for 4 weeks. The dotted line indicates the anastomosis. <b><i>A.</i></b> Images stacked with Z axis up to a total depth of 151 - 201 µm. <b><i>A–a.</i></b> image 26 µm deep to the serosa surface close to the thread around the knot. A small number of neurons are visible.</p

The distribution of total neurons in MOS (n = 5), SB+MOS (n = 4) and vehicle-treated (n = 4) mice.

<p><i>A, B, C.</i> Number of total neurons at depths of every 20 µm. <i>D.</i> Cumulative numbers from all depths.</p

In vivo imaging of enteric neurons in the terminal ileum of an intact Thy1-GFP mouse. <i>A.</i>

<p>28 µm deep to the serosal surface. <b><i>B</i></b>. 34 µm deep to the serosal surface. <b><i>C</i></b>. 42 µm deep to the serosal surface. <b><i>D</i></b>. 60 µm deep to the serosal surface. <b><i>E.</i></b> 125 µm deep to the serosal surface. <b><i>F.</i></b> 145 µm deep to the serosal surface. <b><i>G.</i></b> Merge of 28–50 µm deep images into a single image. Yellow arrows indicate ganglion (ggl) in <b><i>A–C</i></b>, and yellow arrowheads indicate nerve fibers in <b><i>A, B, D</i></b> and <b><i>F</i></b>, respectively. LM: longitudinal muscle. CM: circular muscle. bv: blood vessel. crp: crypt. Cal bar, 100 µm.</p

A stereomicroscopic image including the observed site shown in <b>Figure 4</b>.

<p><b><i>A.</i></b> The thick granulation tissue at the anastomotic region in a mouse that was treated with MOS solution for 1 week after anastomosis surgery. An area in the square (<b>a</b>) corresponds to an area in the square (<b>a</b>) in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054814#pone-0054814-g004" target="_blank"><b>Figure 4</b></a>. <b><i>B.</i></b> A microscopic image of a longitudinal section, prepared following fixation, that was taken along the line (<b>b</b>) indicated in panel <b><i>A</i></b>.</p