High-fat diet-induced alterations to gut microbiota and gut-derived lipoteichoic acid contributes to the development of enteric neuropathy.

BACKGROUND: High-fat diet, microbial alterations and lipopolysaccharide (LPS) are thought to cause enteric diabetic neuropathy and intestinal dysmotility. However, the role of the gut microbiota, lipoteichoic acid (LTA) from Gram-positive bacteria and short-chain fatty acids (SCFAs) in the development of diabetic enteric neuropathy and intestinal dysmotility is not well understood. Our aim was to examine the role of the gut microbiota, LTA and SCFAs in the development of diabetic enteric neuropathy and intestinal dysmotility. METHODS: We fed germ-free (GF) and conventionally raised (CR) mice either a high-fat (HFD) or standard chow diet (SCD) for 8 weeks. We analyzed the microbial community composition in CR mice using 16S rRNA sequencing and damage to myenteric neurons using immunohistochemistry. We also studied the effects of LPS, LTA, and SCFAs on duodenal muscularis externa contractions and myenteric neurons using cultured preparations. KEY RESULTS: High-fat diet ingestion reduced the total number and the number of nitrergic myenteric neurons per ganglion in the duodenum of CR but not in GF-HFD mice. GF mice had fewer neurons per ganglion compared with CR mice. CR mice fed a HFD had increased abundance of Gram-positive bacteria. LTA and LPS did not affect the frequency of duodenal muscularis contractions after 24 hours of cultured but reduced the density of nitrergic myenteric neurons and increased oxidative stress and TNFα production in myenteric ganglia. SCFAs did not affect muscularis contractions or injure myenteric neurons. CONCLUSIONS & INFERENCES: Gut microbial alterations induced increase in Gram-positive bacterial LTA may contribute to enteric neuropathy.

Dangerous Liaison: Helicobacter pylori, Ganglionitis, and Myenteric Gastric Neurons: A Histopathological Study.

Chronic inflammation induced by Helicobacter pylori (H. pylori) infection plays a major role in development of gastric cancer. However, recent findings suggested that progression of inflammation and neoplastic transformation in H. pylori infection are more complex than previously believed and could involve different factors that modulate gastric microenvironment and influence host-pathogen interaction. Among these factors, gastric myenteric plexus and its potential adaptive changes in H. pylori infection received little attention. This study is aimed at identifying the impact of H. pylori-associated gastritis on number and morphology of nerve cells in the stomach. The distribution of density, inflammation, and programmed cell death in neurons was immunohistochemically assessed in full-thickness archival tissue samples obtained from 40 patients with H. pylori infection who underwent surgery for gastric cancer and were compared with findings on samples collected from 40 age- and sex-matched subjects without bacteria. Overall, significant differences were noted between H. pylori-positive and H. pylori-negative patients. The analysis of tissue specimens obtained from those with infection revealed higher density and larger surface of the myenteric nervous plexus, as well as a significant increase in the number of gastric neuronal cell bodies and glial cells compared to controls. A predominant CD3-immunoreactive T cell infiltrate confined to the myenteric plexus was observed in infected subjects. The presence of mature B lymphocytes, plasma cells, and eosinophils was also noted, but to a lesser extent, within the ganglia. Myenteric ganglionitis was associated with degeneration and neuronal loss. Our results represent the first histopathological evidence supporting the hypothesis that H. pylori-induced gastric inflammation may induce morphological changes in myenteric gastric ganglia. These findings could help gain understanding of some still unclear aspects of pathogenesis of H. pylori infection, with the possibility of having broader implications for gastric cancer progression.

Toxoplasma gondii promotes changes in VIPergic submucosal neurons, mucosal intraepithelial lymphocytes, and goblet cells during acute infection in the ileum of rats.

BACKGROUND: The intestinal mucosa plays an important role in the mechanical barrier against pathogens. During Toxoplasma gondii infection, however, the parasites invade the epithelial cells of the small intestine and initiate a local immune response. In the submucosal plexus, this response promotes an imbalance of neurotransmitters and induces neuroplasticity, which can change the integrity of the epithelium and its secretory function. This study evaluated the submucosal neurons throughout acute T. gondii infection and the relationship between possible alterations and the epithelial and immune defense cells of the mucosa. METHODS: Forty Wistar rats were randomly assigned to 8 groups (n = 5): 1 control group, uninfected, and 7 groups infected with an inoculation of 5000 sporulated T. gondii oocysts (ME-49 strain, genotype II). Segments of the ileum were collected for standard histological processing, histochemical techniques, and immunofluorescence. KEY RESULTS: The infection caused progressive neuronal loss in the submucosal general population and changed the proportion of VIPergic neurons throughout the infection periods. These changes may be related to the observed reduction in goblet cells that secret sialomucins and increase in intraepithelial lymphocytes after 24 hours, and the increase in immune cells in the lamina propria after 10 days of infection. The submucosa also presented fibrogenesis, characterizing injury and tissue repair. CONCLUSIONS AND INFERENCES: The acute T. gondii infection in the ileum of rats changes the proportion of VIPergic neurons and the epithelial cells, which can compromise the mucosal defense during infection.

Saccharomyces boulardii CNCM I-745 supplementation reduces gastrointestinal dysfunction in an animal model of IBS.

BACKGROUND: We evaluated the effect of Saccharomyces boulardii CNCM I-745 on intestinal neuromuscular anomalies in an IBS-type mouse model of gastrointestinal motor dysfunctions elicited by Herpes Simplex Virus type 1 (HSV-1) exposure. METHODS: Mice were inoculated intranasally with HSV-1 (102 PFU) or vehicle at time 0 and 4 weeks later by the intragastric (IG) route (108 PFU). Six weeks after IG inoculum, mice were randomly allocated to receive oral gavage with either S. boulardii (107 CFU/day) or vehicle. After 4 weeks the following were determined: a) intestinal motility using fluorescein-isothiocyanate dextran distribution in the gut, fecal pellet expulsion, stool water content, and distal colonic transit of glass beads; b) integrity of the enteric nervous system (ENS) by immunohistochemistry on ileal whole-mount preparations and western blot of protein lysates from ileal longitudinal muscle and myenteric plexus; c) isometric muscle tension with electric field and pharmacological (carbachol) stimulation of ileal segments; and d) intestinal inflammation by levels of tumor necrosis factor α, interleukin(IL)-1ß, IL-10 and IL-4. RESULTS: S. boulardii CNCM I-745 improved HSV-1 induced intestinal dysmotility and alteration of intestinal transit observed ten weeks after IG inoculum of the virus. Also, the probiotic yeast ameliorated the structural alterations of the ENS induced by HSV-1 (i.e., reduced peripherin immunoreactivity and expression, increased glial S100ß protein immunoreactivity and neuronal nitric oxide synthase level, reduced substance P-positive fibers). Moreover, S. boulardii CNCM I-745 diminished the production of HSV-1 associated pro-inflammatory cytokines in the myenteric plexus and increased levels of anti-inflammatory interleukins. CONCLUSIONS: S. boulardii CNCM I-745 ameliorated gastrointestinal neuromuscular anomalies in a mouse model of gut dysfunctions typically observed with irritable bowel syndrome.

Bifidobacterium longum NCC3001 inhibits AH neuron excitability.

BACKGROUND: Bifidobacterium longum (B. longum) NCC3001 can affect behavior and brain biochemistry, but identification of the cellular targets needs further investigation. Our hypothesis was that the communication with the brain might start with action on enteric sensory neurons. METHODS: Ileal segments from adult mice were used to create a longitudinal muscle-myenteric-plexus preparation to expose sensory after-hyperpolarizing (AH) neurons in the myenteric plexus to allow access with microelectrodes. The intrinsic excitability of AH neurons was tested in response to the perfusion of conditioned media (B. longum culture supernatant) or unconditioned media (growth medium, MRS). KEY RESULTS: B. longum conditioned medium significantly reduced the excitability of AH neurons compared to perfusion with the unconditioned medium. Specifically, a reduction was seen in the number of action potentials fired per depolarizing test pulse, the instantaneous and time-dependent input resistances and the magnitude of the hyperpolarization-activated cationic current (Ih ). CONCLUSIONS & INFERENCES: The probiotic B. longum reduces excitability of AH sensory neurons likely via opening of potassium channels and closing of hyperpolarization-activated cation channels.

Consequences of Citrobacter rodentium infection on enteroendocrine cells and the enteric nervous system in the mouse colon.

We tested the hypothesis that Citrobacter rodentium infection leads to changes in the mucosal enteroendocrine signalling and the enteric nervous system and that the host's immune response contributes to these changes. Enteroendocrine cells, serotonin (5-HT) reuptake transporter (SERT), 5-HT release, and inducible nitric oxide synthase (iNOS) expression were assessed in the colon of infected wild-type or severe combined immunodeficient (SCID) mice. Immunoreactivity for iNOS and neuropeptides were examined in the submucosal and myenteric plexuses. Mice were orogastrically infected with C. rodentium and experiments were conducted during the injury phase (10 days) and the recovery phase (30 days). 5-HT and somatostatin enteroendocrine cells and SERT were significantly reduced 10 days after infection, with numbers returning to control values at 30 days. 5-HT release was increased at 10 days. Changes to the mucosal serotonin signalling system were not observed in SCID mice. iNOS immunoreactivity was increased in the submucosa and mucosa at 10 days and returned to baseline levels by 30 days. No differences were observed in neuropeptide or iNOS immunoreactivity in the enteric plexuses following infection. The host's immune response underlies changes to enteroendocrine cells, SERT expression and 5-HT release in C. rodentium infection. These changes could contribute to disturbances in gut function arising from enteric infection.

Effects of the probiotic yeast Saccharomyces boulardii on the neurochemistry of myenteric neurones in pig jejunum.

We studied the effects of food supplementation with Saccharomyces boulardii (S. boulardii; synonym S. cerevisiae HANSEN CBS 5926; 1 g per day for 9 days) on the presence and co-localization patterns of neuronal markers in myenteric neurones of the pig jejunum. The pan neuronal marker Hu revealed no change in the number of neuronal cell bodies per ganglion (37 +/- 7 in control vs 34 +/- 9 in the S. boulardii group). Ranked by size the following cell populations were identified: choline acetyltransferase (ChAT), calbindin-28k (CALB), substance P (SP), neurofilament 160 kD (NF-160), vasoactive intestinal polypeptide (VIP), nitric oxide synthase (NOS), calcitonin gene-related peptide (CGRP), calretinin (CALRET). We found a significant decrease in the number of CALB myenteric neurones in animals which received S. boulardii supplemented diet. None of the other neuronal markers revealed any difference between controls and S. boulardii treated animals. The study reports transmitter-localization patterns in the myenteric plexus of the pig jejunum and provides evidence that changes in the neurochemistry of enteric neurones occur with S. boulardii supplemented diet. Although only CALB expression was altered and the functional significance of this finding remains unknown, our study identified a possible new effector level of probiotics in the gut.

Varicella-zoster virus DNA in the oesophageal myenteric plexus in achalasia.

In a search for past or present infection with herpes viruses, serum antibody titres to herpes simplex type 1 virus, cytomegalovirus, and varicella-zoster virus were measured by complement fixation test in 58 patients with achalasia. Serum was also taken from 40 age and sex matched patients without oesophageal symptoms who formed a control group. All titres were low, and those for herpes simplex type 1 virus and cytomegalovirus did not differ in the achalasia patients and the controls. However, the incidence of varicella-zoster virus antibodies was significantly greater in the achalasia than in the control group (p < 0.05). Using oesophageal tissue containing myenteric plexus removed at the time of cardiomyotomy in nine patients with achalasia, in situ DNA hybridisation showed evidence of varicella-zoster virus in three, but all were negative for the other two viruses. No positive results were obtained for herpes simplex type 1 virus, cytomegalovirus, or varicella-zoster virus in oesophageal tissue from 20 patients undergoing oesophageal resection for diseases other than achalasia. The incidence of positivity for varicella-zoster virus was significantly increased in the achalasia group compared with the controls (p < 0.02). The findings indicate that varicella-zoster virus DNA may persist in the oesophageal myenteric plexus in some patients with achalasia and raise the possibility that this virus is of aetiological importance in achalasia.

Direct spread of reovirus from the intestinal lumen to the central nervous system through vagal autonomic nerve fibers.

A crucial event in the pathogenesis of systemic enteric virus infections is entry of virus into the nervous system. Whether enteric virus spreads from the intestinal tract to the central nervous system through nerves or through the bloodstream was examined using a serotype 3 reovirus strain. After peroral inoculation of newborn mice with reovirus, serial histologic sections of small intestine, brain and spinal cord were prepared and stained by immunoperoxidase to detect viral antigen. Three days after inoculation, viral antigen was observed in mononuclear cells of ileal Peyer's patches and in neurons of the adjacent myenteric plexus. Infection first appeared in the central nervous system 1-2 days later in neurons of the dorsal motor nucleus of the vagus nerve. Endothelial cells, meninges, choroid plexus, hypothalamus, and area postrema were not infected, indicating neural rather than bloodborne spread from the intestine. Staining of neurons in the dorsal motor nucleus of the vagus nerve depended on the route of virus inoculation and was independent of the amount of virus in the bloodstream. These results demonstrate that an enteric virus entering a host from the intestinal lumen can spread to the central nervous system through nerve fiber innervating the intestine.

Spread of herpes simplex virus type-1 (Miyama +GC strain) to the central nervous system after intraperitoneal inoculation: the role of the myenteric plexus of the gut.

The pathways taken by the HSV-1 virus after intraperitoneal (i.p.) inoculation were studied in 5-week old male C3H/HeN mice injected with 1 x 10(4) PFU (100 LD50) or 5 x 10(5) PFU (5000 LD50) of HSV-1 (Miyama +GC strain). At the higher dosage (5 x 10(5) PFU), HSV-1 began replicating in the adrenal from the first day, then in the gut and thoracic portion of the spinal cord by the third day, and in the brainstem by the fourth day, as shown by the titers of the virus in these organs. By immunoperoxidase staining HSV-1 was localized in a necrotic area of the adrenal, the myenteric plexus of the gut, the intermediolateral columns of the thoracic cord, and the vagus nerve nuclei of the medulla oblongata. In the low dose mice (1 x 10(4) PFU), HSV-1 was not isolated from the adrenal or thoracic segment of the spinal cord from the time of inoculation until the time of death. It was, however, isolated from the gut on days 4-6 and from the brainstem by day 5. HSV-1 was never isolated from the blood of either group at any time. The localizations of viral replication suggest that in the mice inoculated with 1 x 10(4) PFU, HSV-1 spreads to the brainstem via the vagal nerves after replication in the myenteric plexus of the gut. In the mice given the higher dose, localizations suggest not only the above route, but also that the virus spread to the intermediolateral columns of the spinal cord after replicating in the adrenal.

Autonomic nervous system involvement in experimental genital infection by herpes simplex virus type 2.

Peroxidase-antiperoxidase technique and histology were employed to elucidate the peripheral routes involved in HSV-2 progression from vagina towards the central nervous system in mice. 12 week-old female Balb/c mice were intravaginally infected with 5 X 10(5)LD50 of HSV-2. Sixty per cent of animals developed vulvovaginitis, perigenital alopecia and hind-limb paresia. Death occurred at 9-11 days post-infection. Colon dilatation and urinary bladder distention were observed in all cases. Complete transversal sections from vulva to kidneys were obtained of each animal, including the spinal cord in situ. Herpes antigen were regularly detected in vulvovaginal epithelium, intramural, perigenital and perivesical small nerves. Besides, their invariable presence in Auerbach's plexus and sympathetic ganglia, strongly suggests preferential autonomic nervous system involvement in the progression of HSV-2 intravaginal infection towards the spinal cord.

Cytomegalovirus inclusion disease. Its occurrence in the myenteric plexus of a renal transplant patient.

Cytomegalovirus (CMV) infected the myenteric plexus of the small and large bowels in an 18-year-old renal transplant patient. This is, to our knowledge, a previously unreported condition. The patient died of a massive gastrointestinal hemorrhage, which possibly resulted from a series of CMV cecal ulcers associated with Candida fungal hyphae and spores.