Vivo in the terminal ileum of an intact Thy1-GFP mouse (Figure 2). The normal mouse gut was rather thin, with a maximal thickness at myenteric ganglion level of 50 mm. Although images stacked with a Z-axis depth up to 50 mm showed low signal/background fluorescence intensity ratio, each 1081537 image at depth of 28 mm?2 mm from serosa showed a single myenteric ganglion and longitudinal circular muscle layers (Figure 2A ). Thicker images at depth of 60, 125 and 145 mm also showed nerve 11089-65-9 web fibers in circular muscle layers, blood vessels and nerve fibers around crypts (Figure 2D ), respectively. Living cells in a given ganglion were clearly observed in merged images at depth of 28 mm?0 mm (Figure 2G). A stereomicroscopic image demonstrating the thick granulation tissue at the anastomotic region in mouse treated with MOS solution 23115181 for 1 week after surgical anastomosis is shown in Figure 3A. However, under a stereomicroscopy no nerve cells or fibers were visible. A longitudinal section including the sameFigure 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 mm. A . image 26 mm deep to the serosa surface close to the thread around the knot. A small number of neurons are visible. doi:10.1371/journal.pone.0054814.ggranulation tissue indicates an obvious thickness of the tissue (Figure 3B). Using confocal imaging of the fixed whole mount preparation, no nerve cells or fibers were visible in the granulation tissue at the anastomosis, although intact myenteric plexus was visible in the intact area in a mouse treated with MOS solution for 1 week after surgery (Figure 4). The living ileum before fixation is shown in Figure 5. In mice treated with MOS solution for 1 week after anastomosis, new ganglia-like structures and nerve fibers wereIn Vivo Imaging of Enteric NeurogenesisFigure 8. The average cell 14636-12-5 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. doi:10.1371/journal.pone.0054814.gobserved by in vivo imaging of the thick granulation tissue at the anastomotic region of the living ileum (Figure 5A). In sites bothoral (Figure 5A -a’) and anal (Figure 5A 2b’) to the anastomotic site, new ganglia-like structures packed with many ofIn Vivo Imaging of Enteric Neurogenesisnewborn neurons and interganglionic nerve fibers were apparent in each image regardless of the depth from the serosa indicating that in the granulation tissue a new enteric neural network was being formed. The density of neurons observed within the anastomosis was 511 cells per 864,900 mm2. The distribution of neurons was even in each nine field (from a21 to c23) (Figure 5B). Figure 5C clearly illustrates new neurons in two typical ganglion-like structures. In mice treated with the 5-HT4 antagonist, SB 207266 (SB) plus MOS solution for 1 week after anastomosis, no neurons or nerve fibers were observed in the anastomotic region (Figure 6), although aggregates of small cells (not neurons) were observed near the surface (Figure 6A ). Thus, enteric neurogenesis was largely suppressed by simultaneous administration of the 5HT4 receptor blocker, SB along with MOS. Similar.Vivo in the terminal ileum of an intact Thy1-GFP mouse (Figure 2). The normal mouse gut was rather thin, with a maximal thickness at myenteric ganglion level of 50 mm. Although images stacked with a Z-axis depth up to 50 mm showed low signal/background fluorescence intensity ratio, each 1081537 image at depth of 28 mm?2 mm from serosa showed a single myenteric ganglion and longitudinal circular muscle layers (Figure 2A ). Thicker images at depth of 60, 125 and 145 mm also showed nerve fibers in circular muscle layers, blood vessels and nerve fibers around crypts (Figure 2D ), respectively. Living cells in a given ganglion were clearly observed in merged images at depth of 28 mm?0 mm (Figure 2G). A stereomicroscopic image demonstrating the thick granulation tissue at the anastomotic region in mouse treated with MOS solution 23115181 for 1 week after surgical anastomosis is shown in Figure 3A. However, under a stereomicroscopy no nerve cells or fibers were visible. A longitudinal section including the sameFigure 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 mm. A . image 26 mm deep to the serosa surface close to the thread around the knot. A small number of neurons are visible. doi:10.1371/journal.pone.0054814.ggranulation tissue indicates an obvious thickness of the tissue (Figure 3B). Using confocal imaging of the fixed whole mount preparation, no nerve cells or fibers were visible in the granulation tissue at the anastomosis, although intact myenteric plexus was visible in the intact area in a mouse treated with MOS solution for 1 week after surgery (Figure 4). The living ileum before fixation is shown in Figure 5. In mice treated with MOS solution for 1 week after anastomosis, new ganglia-like structures and nerve fibers wereIn Vivo Imaging of Enteric NeurogenesisFigure 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. doi:10.1371/journal.pone.0054814.gobserved by in vivo imaging of the thick granulation tissue at the anastomotic region of the living ileum (Figure 5A). In sites bothoral (Figure 5A -a’) and anal (Figure 5A 2b’) to the anastomotic site, new ganglia-like structures packed with many ofIn Vivo Imaging of Enteric Neurogenesisnewborn neurons and interganglionic nerve fibers were apparent in each image regardless of the depth from the serosa indicating that in the granulation tissue a new enteric neural network was being formed. The density of neurons observed within the anastomosis was 511 cells per 864,900 mm2. The distribution of neurons was even in each nine field (from a21 to c23) (Figure 5B). Figure 5C clearly illustrates new neurons in two typical ganglion-like structures. In mice treated with the 5-HT4 antagonist, SB 207266 (SB) plus MOS solution for 1 week after anastomosis, no neurons or nerve fibers were observed in the anastomotic region (Figure 6), although aggregates of small cells (not neurons) were observed near the surface (Figure 6A ). Thus, enteric neurogenesis was largely suppressed by simultaneous administration of the 5HT4 receptor blocker, SB along with MOS. Similar.