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Review
. 2015;51:82-94.
doi: 10.1540/jsmr.51.82.

Activation of 5-HT4 Receptors Facilitates Neurogenesis of Injured Enteric Neurons at an Anastomosis in the Lower Gut

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Free PMC article
Review

Activation of 5-HT4 Receptors Facilitates Neurogenesis of Injured Enteric Neurons at an Anastomosis in the Lower Gut

Miyako Takaki et al. J Smooth Muscle Res. .
Free PMC article

Abstract

Two-photon microscopy (2PM) can enable high-resolution deep imaging of thick tissue by exciting a fluorescent dye and protein at anastomotic sites in the mouse small intestine in vivo. We performed gut surgery and transplanted neural stem cells (NSC) from the embryonic central nervous system after marking them with the fluorescent cell linker, PKH26. We found that neurons differentiated from transplanted NSC (PKH [+]) and newborn enteric neurons differentiated from mobilized (host) NSC (YFP [+]) could be localized within the granulation tissue of anastomoses. A 5-HT4-receptor agonist, mosapride citrate (MOS), significantly increased the number of PKH (+) and YFP (+) neurons by 2.5-fold (P<0.005). The distribution patterns of PKH (+) neurons were similar to those of YFP (+) neurons. On the other hand, the 5-HT4-receptor antagonist, SB-207266 abolished these effects of MOS. These results indicate that neurogenesis from transplanted NSC is facilitated by activation of 5-HT4-receptors. Thus, a combination of drug administration and cell transplantation could be more beneficial than exclusive cell transplantation in treating Hirschsprung's disease and related disorders including post rectal cancer surgery. The underlying mechanisms for its action were explored using immunohistochemistry of the longitudinal mouse ileum and rat rectal preparations including an anastomosis. MOS significantly increased the number of new neurons, but not when co-administered with either of a protein tyrosine kinase receptor, c-RET two inhibitors. The c-RET signaling pathway contributes to enteric neurogenesis facilitated by MOS. In the future, we would perform functional studies of new neurons over the thick granulation tissue at anastomoses, using in vivo imaging with 2PM and double transgenic mice expressing a calcium indicator such as GCaMP6 and channelrhodopsin.

Figures

Fig. 1.
Fig. 1.
Brain-derived neurotrophic factor (BDNF) facilitates c-ret expression in day 4, and an embryoid body (EB) in hanging drop culture and ES gut. Immunoreactivity associated with trkB, c-ret, sox9 and p75, but no NF or c-kit immunoreactivity, was identified after 4 days, treated with BDNF (BDNF+Day 4). No trkB, c-ret, NF, c-kit, sox9 or p75 immunoreactivity was seen after 4 days if not treated with BDNF (BDNF–Day 4). Immunoreactivity associated with trkB and c-ret, but no NF, c-kit, sox9 or p75 immunoreactivity, was identified in an EB treated with BDNF (BDNF+EB). No trkB, c-ret, NF c-kit, sox9 or p75 immunoreactivity was seen in another EB not treated with BDNF (BDNF–EB). C-ret immunoreactivity was apparent in an ES gut differentiated from EB (BDNF+) after 2 weeks in outgrowth culture (BDNF+ES gut). No c-ret immunoreactivity was detected in ES guts differentiated from EB (BDNF–) after 2 weeks in outgrowth culture (BDNF–ES gut). This figure was reproduced and modified from ref. (1) with permission.
Fig. 2.
Fig. 2.
Representative images of immunostaining for tyrosine receptor kinase B (TrkB) in the intact rectum treated with BDNF for 2 weeks (wk) [Intact+BDNF(+)](A) and in the newly formed granulation tissue treated with saline for 2 wk [BDNF(-)2W](B) and treated with BDNF for 2 wk [BDNF(+)2W](C) and treated with BDNF for 4 wk [BDNF(+)4W](D). A: TrkB-positive cells indicated by arrows were observed in myenteric ganglia of the intact rectum. B: TrkB-positive cells were rarely observed in the granulation tissue within the 2-mm rectal anastomotic site. C: TrkB-positive cells were more frequently observed than NF-positive cells in this region. D: Number of TrkB-positive cells decreased. This figure was reproduced and modified from ref. (3).
Fig. 3.
Fig. 3.
Immunohistochemical detection of neurofilaments (NF) in a dome-like structure in the embryonic stem (ES) gut after 14–21 day outgrowth culture differentiated from embryoid bodies (EBs) treated with 1 µmol l−1 MOS. Yellow arrows indicate newly differentiated neurons. White arrows indicate nerve fibers. Not all structures labeled by red color are neurons, but is the result of non-specific binding. This figure was reproduced and modified from ref. (4) with permission.
Fig. 4.
Fig. 4.
Immunoreactivity in the granulation tissue at the anastomosis (A-E) and distribution of total number of new neurons at the anastomosis (F-I). This figure was reproduced and modified from ref. (6). Abbreviations: see ref. (6).
Fig. 5.
Fig. 5.
Two-PM images of anastomotic region in a neural stem cell (NSC)-transplanted and MOS-treated YFP mouse for 2 weeks. PKH26 fluorescence (+) /YFP fluorescence (+) [PKH26 (+)/YFP (+)] neurons were distributed in each 9 field (a-1~c-3; field size: 310 µm × 310 µm) around the knot at the anastomosis. Mid-right areas (b-3) were demonstrated at depths of 107 and 110 µm. Each white arrowhead indicates a nucleus. Orange solid arrows indicated PKH26 (+) neurons and yellow solid arrows indicated YFP (+) neurons (not all). Overlapped neurons between both depths were not counted as indicated by each open arrow. This figure was reproduced and modified from ref. (10).
Fig. 6.
Fig. 6.
Supposed underlying mechanism for neurogenesis from transplant neural stem cells (NSC) and host NSC at the anastomosis. SDF-1/CXCL12: chemokine stromal cell-derived factor-1; GDNF: glia-derived neurotrophic factor; GFRα: GDNF family receptor α.
Fig. 7.
Fig. 7.
Molecular mechanism for neurogenesis activated by 5-HT4 receptors by MOS. Main intracellular pathways activated by proto-oncogene tyrosine-protein kinase receptor Ret (RET) are shown. MOS activates a 5-HT4 receptor (SR4), G protein coupled receptor (GPCR). This GPCR possibly does transactivation of RET. RET activation results in phosphorylation of several residues, including Y1015 and Y1062. Growth factor receptor-bound protein 2 (GRB2) and phospholipase C gamma (PLCγ) are required for proliferation and/or differentiation of enteric nervous system (ENS) precursors. RAC, RHO and CDC42 regulate enteric neural crest-derived cells (ENCDCs) migration and proliferation. Kinesin-like protein KIF26A (KIF26A), Sprouty2 (SPRY2) and phosphatase and tensin homolog (PTEN) are negative regulators of RET signalling: KIF26A binds to GRB2 and prevents RAS-ERK and phosphoinositide 3-kinase (PI3 K) signalling, whereas SPRY2 binds to RAS and RAF and blocks activation of the RAS-ERK pathway. PTEN regulates proliferation via PI3 K and Akt. Retinoic acid (RA) reduces PTEN levels in migrating ENCDCs and hence blocks the inhibitory effect of PTEN on RET signalling. Two circled pathways supposed to be more important than others. This figure was reproduced and modified from ref. (22) and ref. (26) with permission. GAB1: GRB2-associated binding protein 1; GDNF: glial cell line-derived neurotrophic factor; GFRα1: GDNF family receptor α1; GRB2: growth factor receptor-bound protein 2; JNK: c-Jun-NH2-terminal kinase; PKC: protein kinase C; S: serine; Y: tyrosine.
Fig. 8.
Fig. 8.
Perspectives: Functional studies of new born neurons at the anastomosis.

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