Enteroendocrine cells and 5-HT availability are altered in mucosa of guinea pigs with TNBS ileitis

Enteroendocrine cells act as sensory transducers, releasing 5-HT and numerous peptides that are involved in regulating motility, secretion, and gut sensation. The action of mucosal 5-HT is terminated by a 5-HT reuptake transporter (SERT). In this study, we examined the hypothesis that ileitis leads to changes in enteroendocrine cell populations and mucosal 5-HT availability. Ileitis was induced in guinea pigs by intraluminal injection of 2,4,6-trinitrobenzenesulfonic acid and experiments were conducted 3, 7, and 14 days after treatment. The number of somatostatin, neurotensin, and 5-HT-immunoreactive cells increased at 3 and 7 days of ileitis, respectively, whereas no significant changes in the numbers of cholecystokinin, glucagon-like peptide-2, glucose-dependent insulinotropic peptide, and peptide YY-immunoreactive cells were observed. Chemical stimulation of the inflamed mucosa with sodium deoxycholic acid significantly increased 5-HT release compared with basal release. Mechanical stimulation of the mucosa potentiated the effect of the chemical stimuli at day 7. Epithelial SERT immunoreactivity was significantly reduced during the time course of inflammation. Thus changes in enteroendocrine cell populations and 5-HT availability could contribute to the altered motility and secretion associated with intestinal inflammation by disrupting mucosal signaling to enteric nerves involved in peristaltic and secretory reflexes.     

Olfactory mucosa response in guinea pigs following intranasal instillation withCryptococcus neoformans

A central nervous system isolate from an acquired immunodeficiency syndrome (AIDS) patient of 10³ Cryptococcus neoformans cells was instilled intranasally into guinea pigs. These were killed to evaluate the fate of the organisms and the response of the olfactory mucosa. Olfactory epithelium prevented the penetration ofCryptococcus neoformans and showed focal hyperplastic responses. The organisms, which manifested an affinity for the olfactory portion of the nasal cavities, were cleared from the olfactory space with no intervention from the immune system cells. By the end of the fifth week almost no organisms could be found and there was no histological evidence of dissemination. In contrast, destruction of the olfactory epithelium with zinc sulfate allowed the invasion of the subepithelial tissues, demonstrating the role of the olfactory mucosa in preventing infection withCryptococcus neoformans through the nasal route. The results and the model described in this report may be useful in clarifying the pathogenic mechanisms of cryptococcosis and the non immune mediated host responses toCryptococcus neoformans.     

BMP signaling in rats with TNBS-induced colitis following BMP7 therapy

Beyond stimulating bone formation, bone morphogenetic proteins (BMPs) are important in development, inflammation, and malignancy of the gut. We have previously shown that BMP7 has a regenerative, anti-inflammatory, and antiproliferative effect on experimental inflammatory bowel disease (IBD) in rats. To further investigate the BMP signaling pathway we monitored the effect of BMP7 therapy on the BMP signaling components in the rat colon during different stages of experimentally induced colitis by 2,4,6-trinitrobenzene sulfonic acid (TNBS). The results showed a significantly decreased BMP7 expression in the acute phase, followed by a significantly increased BMP2 and decreased BMP6 expression during the chronic phase of colitis. BMP7 therapy influenced the expression of several BMPs with the most prominent effect on downregulation of BMP2 and upregulation of BMP4 in the chronic phase of colitis. Importantly, connective tissue growth factor and noggin expression were elevated in the acute stage and significantly decreased upon BMP7 therapy. BMP receptor I expression was unchanged, whereas BMP receptor II was decreased at day 2 and increased at days 14 and 30 of TNBS inflammation. However, an opposite pattern of expression following BMP7 therapy has been observed. BMP7 increased the expression of BR-Smad including Smad3 and Smad4. Inhibitory Smads were increased in colitis and significantly decreased following BMP7 therapy at later stages of the disease. We suggest that BMP signaling was altered during TNBS-induced colitis and was recovered with BMP7 administration, suggesting that IBD is a reversible process.     

Quantitative analysis of squamous epithelium of normal palatal mucosa in guinea pigs

Summary The epithelium of intact guinea pig palate was subjected to stereologic analysis in a study of structural alterations in the keratinizing epithelium in response to wounding. Point counting procedures were employed to analyse electron micrographs sampled from three epithelial strata in biopsies collected from five animals. The differentiation pattern of the guinea pig palate epithelium displayed the following structural density gradients from basal to granular layers: descending gradients of metabolically active organelles, ascending gradient of bundled filaments coupled with the appearence of membrane coating granules and keratohyalin granules, and a plateau-like gradient of cytoplasmic ground substance. This pattern of epithelial differentiation is basically identical to that of human hard palate epithelium and epidermis. Regional and species variations in structure of keratinizing epithelia are suggested based on interepithelial differences in morphometric parameters.This investigation was supported in part by grant No. 512-4064 from the Danish State Medical Research Council and by a grant from the Calcin Foundation.The data recording and computation was performed on a guest visit at the Dental Institute, University of Zürich.     

Effect of foreign bodies on the gallbladder mucosa of guinea pigs

Foreign bodies were introduced in the gallbladder of the guinea pig in order to study their effect on the cell proliferative parameters in the gallbladder mucosa. In vitro labeling with radioactive thymidine and autoradiographies were used. Our data show that the surgical manipulation of the control gallbladders had no effect on the mucosa. The introduction of polystyrene bodies was accompanied by a significant increase in DNA synthesis activity in the gallbladder epithelium.     

Chronic administration of galanin attenuates the TNBS-induced colitis in rats

Inflammatory bowel disease (IBD) is a chronic intestinal inflammatory disorder considered as a consequence of an aberrant response of the immune system to luminal antigens. Numerous groups of agents are being evaluated as novel therapeutic approaches for its treatment; in this way, different peptides have emerged as potential candidates. Galanin is an active neuropeptide distributed in the central and periphery nervous systems although it has been also described having important autocrine and paracrine regulatory capacities with interesting inflammatory and immune properties. In this line, we have observed that galanin treatment has a significant preventive effect in the experimental trinitrobenzensulfonic acid (TNBS) acute model of inflammatory colitis. The aim of the present study was to investigate intensively the role played by the peptide in the evolution of the inflammatory pathology associated to IBD. Galanin (5 and 10 microg/kg/day) was administered i.p., daily, starting 24 h after TNBS instillation, and continuing for 14 and 21 days. The lesions were blindly scored according to macroscopic and histological analyses and quantified as ulcer index. The results demonstrated that chronic administration of galanin improved the colon injury than the TNBS induced. The study by Western-blotting of the expression of nitric oxide inducible enzyme (iNOS), as well as the total nitrite production (NO) assayed by Griess-reaction, showed significant reduction associated with peptide administration. The number of mast cells was also identified in histological preparations stained with toluidine blue and the results showed that samples from galanin treatment, mostly at 21 days, had increased the number of these cells and many of them had a degranulated feature. In conclusion, chronic administration of galanin is able to exert a beneficial effect in the animal model of IBD assayed improving the reparative process. Participation of nitric oxide pathways and mucosal mast cells can not be discarded.     

Vacuolating cytotoxin A is associated with increased thrombin generation in gastric mucosa

BACKGROUND: Activation of the coagulation system is a critical response for both the repair of tissue injury and the host defense against microbial pathogens. Activation of the coagulation cascade culminates with the generation of thrombin. In vitro studies have shown that thrombin protects gastric epithelial cells from injury. The present study was undertaken to assess in vivo the relationship between gastric intramucosal generation of thrombin and Helicobacter pylori infection. MATERIALS AND METHODS: This study comprised 59 patients with gastroduodenal disorders. There were 27 patients with H. pylori infection (Hp+), 14 without it (Hp-), and 18 patients with cured H. pylori infection (Hp c). The gastric intramucosal concentrations of thrombin-antithrombin complex (TAT), epidermal growth factor (EGF), prostaglandin E2 (PGE2), and vacuolating cytotoxin A (VacA) were measured by specific immunoassays. RESULTS: The level of TAT was significantly increased in patients with Hp+ compared to Hp- and Hp c. The levels of TAT, EGF and PGE2 were higher in VacA (+) patients than in those with VacA (-). VacA induced significant expression of tissue factor in gastric epithelial cells in vitro. The gastric intramucosal level of VacA antigen was proportionally and significantly correlated with TAT, EGF and PGE2 in Hp+ patients. The level of TAT was proportionally and significantly correlated with EGF in Hp+ patients but not in Hp- and HP c patients. CONCLUSIONS: These results showed that VacA produced by H. pylori is associated with increased thrombin generation, and that thrombin may play a protective role in H. pylori-associated gastroduodenal disorders.     

Aquaporin 3 and 8 are down-regulated in TNBS-induced rat colitis

Aquaporins (AQPs) plays an important role in transcellular water movement, but the AQPs expression profile has not been demonstrated in 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis which closely mimics human Crohn’s disease (CD) histopathologically. To solve the problem, 30 female Sprague–Dawley (SD) rats were randomly divided into a model group (n = 18), an ethanol control group (n = 6) and a normal control group (n = 6). On day 1, the rats in the model group received TNBS + 50% ethanol via the rectum, while the ethanol control rats received an equal volume of 50% ethanol and the normal control rats did not receive any treatment. All rats were sacrificed on day 7, and ileum, proximal colon and distal colon specimens were obtained to examine the alteration in AQP3 and AQP8 using real-time polymerase chain reaction, Western blot analysis and immunohistochemistry. As a result, exposure to TNBS + ethanol resulted in a marked decrease in both the mRNA and protein expression of AQP3 and AQP8, with the exception of AQP8 protein which was negative in the distal colon in all three groups. These reductions in AQP3 and AQP8 were accompanied by an increase in intestinal inflammation and injury. The results obtained here implied that both AQP3 and AQP8 may be involved in the pathogenesis of inflammatory bowel disease.     

Quantitative analysis of epithelial changes during wound healing in palatal mucosa of guinea pigs

Summary The epithelium of wounded guinea pig palate was subjected to stereologic analysis. A total of 18 biopsies (animals) were used. Biopsies were taken at 18, 48, 96 and 120 h after wounding. Point counting procedures were employed to analyse electron micrographs sampled from one (18 h) or two epithelial strata (48, 96 and 120 h). The essential modulations in epithelial structure as wound healing proceeds were as follows: During the early phases characterized by formation and advancement of epithelial lips (18 and 48 h), migrating cells converged towards a cell type which structurally was less differentiated than normal basal cells. This alteration was expressed by a decrease in volume density of cytoplasmic organelles, mainly mitochondria, free ribosomes and tonofilament bundles, coupled with an increase in volume density of lysosomal bodies. Concomitantly, the volume density of cytoplasmic ground substance rose markedly. Subsequent to fusion of contralateral migratory lips (96 and 120 h) reversion to normal epithelial structure was indicated by the increment in magnitude of basal cell parameters. Further structural density gradients from basal towards upper cell layers appeared. This pattern was mainly displayed by mitochondria, free ribosomes, and tonofilament bundles. The magnitude and gradation of most tissue and cell parameters were not yet re-established at 120 h. The density of tonofilament bundles and the density level of cytoplasmic ground substance in particular deviated.This investigation was supported in part by grants No. 512-5958 and No. 512-5151 from the Danish State Medical Research Council     

Effect of pregnant mare's serum gonadotropin on increased ovulation in guinea pigs with synchronized estrous cycle

An ability of Pregnant Mare's Serum Gonadotropin (PMSG) to induce superovulation was investigated in guinea pigs with synchronized estrous cycle caused by the treatment for 21 days of progesterone tubing. On day 6 later following the removal of progesterone treatment, every animal given saline injection had synchronously ovulated. When compared with saline control, a significant increase of ova ovulated was induced by an injection of PMSG 8 hours before the removal of progesterone tubing, but not by the other PMSG treatment schedule. Present study indicates that PMSG injection given at a fixed stage of synchronized estrous cycle induced superovulation in guinea pigs treated with long-term implantation of progesterone tubing.     

Enzymes of glycerolipid synthesis in small-intestinal mucosa of foetal and neonatal guinea pigs.

1. The activities of some enzymes of glycerolipid synthesis were measured in homogenates obtained from the intestinal scrapings of 62-66-day foetuses and 2- and 8-day-old guinea pigs. 2. The ratio of protein concentration/DNA concentration was significantly higher (P greater than 0.001) in homogenized tissue from the neonatal compared with the foetal guinea pigs. Enzyme activities were therefore expressed relative to both protein and to DNA. 3. The specific activities (relative to DNA) of palmitoyl-CoA synthetase, glycerol phosphate acyltransferase and phosphatidate phosphatase were higher in homogenized tissues from neonatal than in those from the foetal guinea pigs. These activities are probably involved more in cell proliferation than in the absorption and transport of triacylglycerol. Its activity was not significantly different in the foetal and neonatal guinea pigs when expressed relative to DNA but it was lower in the neonatal guinea pigs when expressed relative to protein. The entry of food into the intestine after birth is therefore not necessary for its activity.     

Serotonin availability in rat colon is reduced during a Western diet model of obesity

Constipation and slowed transit are associated with diet-induced obesity, although the mechanisms by which this occurs are unclear. Enterochromaffin (EC) cells within the intestinal epithelium respond to mechanical stimulation with the release of serotonin [5-hydroxytryptamine (5-HT)], which promotes transit. Thus our aim was to characterize 5-HT availability in the rat colon of a physiologically relevant model of diet-induced obesity. EC cell numbers were determined immunohistochemically in chow-fed (CF) and Western diet-fed (WD) rats, while electrochemical methods were used to measure mechanically evoked (peak) and steady-state (SS) 5-HT levels. Fluoxetine was used to block the 5-HT reuptake transporter (SERT), and the levels of mRNA for tryptophan hydroxylase 1 and SERT were determined by quantitative PCR, and SERT protein was determined by Western blot. In WD rats, there was a significant decrease in the total number of EC cells per crypt (0.86 ± 0.06 and 0.71 ± 0.05 in CF and WD, respectively), which was supported by a reduction in the levels of 5-HT in WD rats (2.9 ± 1.0 and 10.5 ± 2.6 μM at SS and peak, respectively) compared with CF rats (7.3 ± 0.4 and 18.4 ± 3.4 μM at SS and peak, respectively). SERT-dependent uptake of 5-HT was unchanged, which was supported by a lack of change in SERT protein levels. In WD rats, there was no change in tryptophan hydroxylase 1 mRNA but an increase in SERT mRNA. In conclusion, our data show that foods typical of a WD are associated with decreased 5-HT availability in rat colon. Decreased 5-HT availability is driven primarily by a reduction in the numbers and/or 5-HT content of EC cells, which are likely to be associated with decreased intestinal motility in vivo.     

Effect of COX-2 inhibitor after TNBS-induced colitis in wistar rats

Inflammatory bowel disease (IBD) is a common chronic gastrointestinal disorder characterized by alternating periods of remission and active intestinal inflammation. Some studies suggest that antiinflammatory drugs are a promising alternative for treatment of the disease. Thus, this study aimed to evaluate the effect of lumiracoxib, a selective-cyclooxygenase-2 (COX-2) inhibitor, on 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced experimental colitis. Wistar rats (n = 25) were randomized into four groups, as follows: Group (1) Sham group: sham induced-colitis rats; Group (2) TNBS group: nontreated induced-colitis rats; Group (3) Lumiracoxib control group; and Group (4) Lumiracoxib-treated induced-colitis rats. Our results showed that rats from groups 2 and 4 presented similar histopathological damage and macroscopic injury in the distal colon as depicted by significant statistically differences (P < 0.01; P < 0.05) compared to the other two groups. Weak expression of COX-2 mRNA was detected in normal colon cells, while higher levels of COX-2 mRNA were detected in group 2 and group 4. Therapy with lumiracoxib reduced COX-2 expression by 20–30%, but it was still higher and statistically significant compared to data obtained from the lumiracoxib control group. Treatment with the selective COX-2 inhibitor lumiracoxib did not reduce inflammation-associated colonic injury in TNBS-induced experimental colitis. Thus, the use of COX-2 inhibitors for treating IBD should be considered with caution and warrants further experimental investigation to elucidate their applicability.     

Leishmania braziliensis spp. in the nasal mucosa of guinea pigs inoculated in the tarsi.

Two lots of 20 young male guinea pigs were inoculated subcutaneously in the tarsi with 10(4) amastigotes of Leishmania braziliensis braziliensis or L. b. guyanensis to study the susceptibility of this Neotropical hystricomorph rodent the autochthonous parasites. Almost 50% of the animals showed lesions in the inoculation site and had parasitizations that were infective to hamsters, as shown by inoculating homogenates of the dermal lesion, of the spleen, of the liver, and of the nasal mucosa into hamsters at 20, 40, 60, and 120 days after inoculation of the guinea pig. Smears of the above organs showed the presence of amastigotes. Parasites inoculated into the tarsi were detected early in the skin, spleen, and liver of the guinea pig host. Blood cultures made by cardiopuncture on sacrifice of the guinea pigs were uniformly negative. The nasal mucosa of nearly all animals positive in the skin or viscera was invaded early by the parasites, although with greater frequency between 60 and 120 days post-inoculation. The use of this model for the study of mucocutaneous parasitism by L. braziliensis is discussed, together with the phenomena of parasitism at a distance from the inoculation site, the temperature of the body regions affected, and the possible genetic influence on susceptibility of the guinea pig to L. braziliensis.