Involvement of Enteric Glia in Small Intestine Neuromuscular Dysfunction of Toll-Like Receptor 4-Deficient Mice

Abstract

Enteric glial cells (EGCs) influence nitric oxide (NO)− and adenosine diphosphate (ADP)− mediated signaling in the enteric nervous system (ENS). Since Toll-like receptor 4 (TLR4) participates to EGC homoeostasis, this study aimed to evaluate the possible involvement of EGCs in the alterations of the inhibitory neurotransmission in TLR4−/− mice. Ileal segments from male TLR4−/− and wild-type (WT) C57BL/6J mice were incubated with the gliotoxin fluoroacetate (FA). Alterations in ENS morphology and neurochemical coding were investigated by immunohistochemistry whereas neuromuscular responses were determined by recording non-adrenergic non-cholinergic (NANC) relaxations in isometrically suspended isolated ileal preparations. TLR4−/− ileal segments showed increased iNOS immunoreactivity associated with enhanced NANC relaxation, mediated by iNOS-derived NO and sensitive to P2Y1 inhibition. Treatment with FA diminished iNOS immunoreactivity and partially abolished NO− and ADP− mediated relaxation in the TLR4−/− mouse ileum, with no changes of P2Y1 and connexin-43 immunofluorescence distribution in the ENS. After FA treatment, S100β and GFAP immunoreactivity in TLR4−/− myenteric plexus was reduced to levels comparable to those observed in WT. Our findings show the involvement of EGCs in the alterations of ENS architecture and in the increased purinergic and nitrergic-mediated relaxation, determining gut dysmotility in TLR4−/− mice.

Glial A2B Adenosine Receptors Modulate Abnormal Tachykininergic Responses and Prevent Enteric Inflammation Associated with High Fat Diet-Induced Obesity

“…Our results in SD mice showed that A 2B Rs are expressed mainly in glial cells, and that the FC-mediated inhibition of glial activity also determined an increased staining in neurons, suggesting a possible involvement of A 2B Rs in the glial-neuronal crosstalk at the level of the ENS [21], as previously determined in the central nervous system (CNS), where they appear to be involved in the regulation of astrocyte-derived BDNF (brain-derived neurotropic factor) production [38]. An increase in EGC activity is usually defined as reactive gliosis, a process denoting a broad spectrum of potential responses of glial cells to ENS insults [9,24]. HFD affected both S100β and A 2B R immunoreactivity, determining a marked increment of both antigens in the colonic ENS, suggesting a possible link between reactive gliosis and A 2B R-mediated responses.…”

“…Fresh isolated distal colonic 3 cm segments from SD and HFD mice were gently flushed with warm (37 • C) Krebs solution to remove any luminal content, and incubated with or without 50 µM FC for 1 h in organ baths containing Krebs solution at 37 • C, bubbled with 95% O 2 + 5% CO 2 [9,24]. The colonic segments were then fixed with 4% paraformaldehyde in phosphate buffered saline (PBS) for 2 h at room temperature.…”

“…The colonic segments were then fixed with 4% paraformaldehyde in phosphate buffered saline (PBS) for 2 h at room temperature. After 3 × 15 min washes in PBS, the colonic segments were cut in 0.5 cm pieces, and opened along the mesenteric border to obtain preparations consisting of the longitudinal smooth muscle with attached myenteric plexus (LMMP), as previously described [24]. The colonic LMMP preparations were gently stretched and pinned down on a wax support, permeabilized in PBS with 0.3% Triton X-100 (PBT), and blocked with 2% bovine serum albumin (BSA) dissolved in PBT for 1 h at room temperature [24,25].…”

“…After 3 × 15 min washes in PBS, the colonic segments were cut in 0.5 cm pieces, and opened along the mesenteric border to obtain preparations consisting of the longitudinal smooth muscle with attached myenteric plexus (LMMP), as previously described [24]. The colonic LMMP preparations were gently stretched and pinned down on a wax support, permeabilized in PBS with 0.3% Triton X-100 (PBT), and blocked with 2% bovine serum albumin (BSA) dissolved in PBT for 1 h at room temperature [24,25]. The LMMP preparations were then incubated overnight at room temperature with guinea pig polyclonal anti-rat S100β (1:100; Synaptic Systems, Göttingen, Germany), rabbit polyclonal anti-human A 2B R (1:50; Merck Life Science, Milan, Italy), and mouse biotin-conjugated anti-human-HuC/D (1:100; Thermo Fisher Scientific, Milan, Italy) primary antibodies.…”

“…Fluorescence intensity (density index) of S100β and A 2B R was assessed for each antigen by capturing 20 images per mouse. The intensity of staining for each antibody was expressed as the density index of labelling per myenteric ganglion area, as previously described [24], and was reported as mean ± SEM.…”