Figure 1.
Depiction of the experimental approach for in vivo imaging of the intestine.
Figure 2.
In vivo imaging of enteric neurons in the terminal ileum of an intact Thy1-GFP mouse. A.
28 µm deep to the serosal surface. B. 34 µm deep to the serosal surface. C. 42 µm deep to the serosal surface. D. 60 µm deep to the serosal surface. E. 125 µm deep to the serosal surface. F. 145 µm deep to the serosal surface. G. Merge of 28–50 µm deep images into a single image. Yellow arrows indicate ganglion (ggl) in A–C, and yellow arrowheads indicate nerve fibers in A, B, D and F, respectively. LM: longitudinal muscle. CM: circular muscle. bv: blood vessel. crp: crypt. Cal bar, 100 µm.
Figure 3.
A stereomicroscopic image including the observed site shown in Figure 4.
A. 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 (a) corresponds to an area in the square (a) in Figure 4. B. A microscopic image of a longitudinal section, prepared following fixation, that was taken along the line (b) indicated in panel A.
Figure 4.
Immunohistochemical image for anti-neurofilament (NF) antibody of a whole mount preparation of the same intestine shown in Figure 5.
A–a corresponds to Figure 5A (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.
Figure 5.
Images of anastomotic region of the terminal ileum in a MOS-treated mouse.
The dotted lines indicates the anastomosis site. Around the knot of thread we obtained each image from 9 visual fields. A. Images stacked with Z axis to a total depth of 200–300 µm. A–a. image 42 µm deep to the serosa surface in area (a) in A. A–a’. image 174 µm deep to the serosa surface in the same area (a) in A. A–b. 44 µm deep to the serosa surface in area (b) in A. A–b’. image 101 µm deep to the serosa surface in the same area (b) in A. Arrows indicate nerve cells in A–a’, b and b’, and arrowheads indicate nerve fibers in A–a, a’, b and b’, and circles indicate ganglion-like clusters of neurons in A–a, b and b’, respectively. B. Number of neurons in each field (size: 310 µm×310 µm) around the knot. C. Newborn nerve cells formed ganglion structures indicated by circles. These were enlarged from the images shown in A–b’–i and –ii.
Figure 6.
Images of anastomosis of the ileum in an SB-207266 (SB) plus MOS treated mouse.
SB plus MOS treatment was performed for one week. A. Images stacked in the Z axis with a total depth of 200 - 300 µm. A–a. image 38 µm deep to the serosa surface in area (a) in A. A–b. image 71 µm deep to the serosa surface in area (b) in A. Circles indicate aggregates of small non-neuronal cells (A–a and b), respectively.
Figure 7.
Images of around the suture knot at anastomosis of the ileum in DMSO treated mouse.
Vehicle treatments were performed for 4 weeks. The dotted line indicates the anastomosis. A. Images stacked with Z axis up to a total depth of 151 - 201 µm. A–a. image 26 µm deep to the serosa surface close to the thread around the knot. A small number of neurons are visible.
Figure 8.
The average cell numbers in each of nine fields (A) and all fields (B) at the anastomosis.
The comparison was performed among MOS (n = 5), SB+MOS (n = 4) and vehicle-treated (n = 4) mice. Each of the nine fields corresponds to that in Figure 5B. *, P<0.05 vs. SB+MOS and vehicle. #, P<0.05 vs. vehicle.
Figure 9.
The distribution of total neurons in MOS (n = 5), SB+MOS (n = 4) and vehicle-treated (n = 4) mice.
A, B, C. Number of total neurons at depths of every 20 µm. D. Cumulative numbers from all depths.
Figure 10.
Correctly identified fluorescent neurons by 2PM are proved to be neurons at the anastomosis in MOS-treated mice.
A. Green Fluorescent Protein (GFP)-positive cells. B. 5-bromo-2'-deoxyuridine (BrdU)-positive cells. C. A neural marker, neurofilament (NF)-positive cell. D. A neural stem cell marker, distal less homeobox 2 (DLX2)-positive cells. E. glial fibrillary acidic protein (GFAP)-negative cells. Red arrows indicate NF+/DLX2−/BrdU+/GFP+/GFAP- cell: this cell is a new neuron. Green arrows indicate NF−/DLX2+/BrdU+/GFP+/GFAP- cells: these cells seem to be neural progenitors. Similar results are obtained in other preparations.