Effect of ingestion of green seaweed, Ulva lactuca, (70 g kg−1) during a 6-week period on caecal and colonic mucosas was studied in germ-free (GF) rats and in heteroxenic (HE) rats harbouring a human bacterial flora (GF rats associated with a human flora). The pH and sulphide concentration of the caecal contents, crypt morphometry, mitotic index and mucin types in the caecal and distal colonic mucosas were determined. In the GF caecum, Ulva strongly increased crypt depth and mucin-containing cells irrespective of the mucin type studied (neutral, acidic or sulphated) compared to the control diet but had no significant effect on mitotic index. The crypt depth and mucin-containing cells in the caecum were higher in HE than in GF control rats. They were slightly but significantly increased by Ulva. In the distal colon mucosa of GF rats, Ulva decreased crypt depth and cell number as well as sulphomucin-containing cells. Conversely, in the HE rats, it increased crypt depth and reduced the number of neutral mucin-containing cells. These results show that Ulva lactuca exerts an intrinsic effect on mucosal morphometry and on mucin biosynthesis in GF rats. No pathological alteration was observed in the mucosas and no significant modification of the mitotic index or sulphide production was observed in HE rats. © 1999 Society of Chemical Industry

ABSTRACT Germfree suckling rats were infected with an SA11 rotavirus strain. Infected pups developed diarrhea associated with histopathological changes. The virus was detected in feces and in the small intestine. Cellular vacuolation was observed in the villi of the jejunum. These results provide a new model for further investigations of group A rotavirus infection.

The model of the isolated, vascularly perfused rat colon was assessed in the present study to investigate the nervous, hormonal, and local/paracrine pathways involved in colonic mucin secretion. A colonic loop was perfused via the superior mesenteric artery with a Krebs-Henseleit buffer containing 25% washed bovine erythrocytes at a rate of 2.5 ml/min. After a 10-min control period, each compound to be tested was infused intra-arterially for 30 min. Tissue samples from the proximal and midsegments of the perfused rat colon were then fixed and stained for mucus cell count. Intra-arterial administration of bethanechol evoked a concentration-dependent decrease in the number of stained mucus cells per crypt section over the range 2.10(-6) to 2.10(-4) M: 16.6 +/- 1.4 stained mucus cells per crypt in the midportion of the perfused rat colon (n = 5) with bethanechol 2.10(-4) M versus 28.8 +/- 1.5 for controls (n = 6). After infusion of 1.25 and 2.5 mu M 16,16-dimethyl prostaglandin E-2 (dmPGE(2)), the number of stained mucus cells per crypt section was significantly reduced: 21.6 +/- 0.6 (n = 6) and 20.6 +/- 1.4 (n = 7), respectively. An increase in the number of cavitated mucus cells was also observed (22.1 -/+ 6.7 and 38.5 +/- 4.1% of cavitated mucus cells in the midsegment of the perfused rat colon with 1.25 and 2.5 mu M dmPGE(2), respectively, vs. 12.3 +/- 4.1% for controls). In contrast, prostaglandin F-2 alpha did not significantly affect mucus discharge from colonic cells. Peptide YY (10(-10), 10(-9) and 10(-8) M) induced a dose-dependent increase in the percentage of cavitated mucus cells (16.7 +/- 2.8, 23.1 +/- 4.2, and 31.2 +/- 3.4% of cavitated mucus cells in the midsegment, respectively). The proximal and midsegments of the perfused rat colon were equally sensitive to each secretagogue.Conclusion: In the isolated, vascularly perfused rat colon, mucus cells strongly respond to the well-known mucin secretagogues, bethanechol and dmPGE(2). This approach has already led to the identification of a novel stimulant of mucin secretion: peptide YY. Our ex vivo model, in which goblet cells are submitted to well-defined luminal and blood-borne stimuli is, therefore, reliable to investigate the nervous, hormonal, and local/paracrine pathways involved in the colonic mucin secretion.

A colorimetric method was used on water-soluble much extracted from mucosal scrapings and contents of the caecum and the colon of five germ-free (GF) rats and five heteroxenic (HE) rats harbouring a hnmao flora (GF rats d a t e d with a human flora). These rats were fed on a diet containing either 100 g sucrose/kg or 100 g inulin/kg. Histological stains, periodic acid–Schiff, alcian blue pH 2·5 and alcian blue pH 0·5 were used to discriminate between neutral, acidic and acidic sulphated mucins respectively. Spectrocolorimetric assays led to a calculated absorbance value for 1 mg of the initial much extract. Each much type was compared between treatments. The caecal contents of GF rats contained more acidic mucin than sulphomucin, which was present in the same proportion as neutral mucin. Their colonic contents contained more acidic mucins than sulphomucin, which in turn was more abundant than neutral mucin. Their caecal mucosa mucin distribution differed from that of the contents: very little acidic much was present and neutral and dphomucin proportions were of the same order of magnitude. Inulin increased the amount of neutral much in the caecal contents and of sulphated mucios in the colonic contents and increased the amounts of neutral and acidic mucins in the caecal mucwa. Mucin distribution in the HE rats was very different from that in the GF rats: the caecal contents contained a high proportion of acidic much and very little sulphomucin. The same distribution of mucins was observed in the colonic contents. The caecal mucaw contained less acidic much and more sulphomucin than the caecal contents. Inulin decreased acidic mucins and increased sulphated much in the caecal contents and increased neutral and sulphated mucins in the colonic contents. Inulin increased sulphomucin in the caecal m u m and decreased acidic much in the caecal and colonic mucosas. The very low amount of mucin that was recovered in the colonic mucosa suggests that, in the presence of the bacterial flora and associated with inulin in the diet, much was extensively released from the mucosa to the colonic lumen. This might be related to the bacterial metabolites produced

A method, transposed from the mucin histochemical stainings, was proposed to evaluate neutral, acidic and sulphated mucins by spectrocolorimetry. Stainings used were periodic acid/Schiff (PAS), Alcian blue (AB) pH 2.5, and Alcian blue (AB) pH 0.5. Mucin samples were extracted from mucosal scrapings, from intestinal contents of germ-free rats or from commercial pig gastric mucin. A mucin-enriched fraction was obtained and lyophilised and the protein content was determined. Each mucin type was spectrophotometrically analysed after staining and precipitation by slightly modified Carnoy fixative. The histochemical stainings used here, after modification by Carnoy fixative treatment, were, as shown by electrophoretic controls, specific for mucin types contained in these mucins without protein contaminant interference.

ABSTRACT: In the pig, the gastrointestinal tract grows rapidly after birth and undergoes a short postnatal maturation. The objective of the present work was to assess the metabolic characteristics of the small intestinal mucosa during this period by investigating glucose, galactose, and glutamine metabolism in pig isolated enterocytes. Piglets were used immediately after birth or at various stages during suckling or postweaning. Fed animals were taken in a postabsorptive state. The jejunoileum was excised and perfused with an EDTA (5 mM)-containing buffer. The epithelial cell layer was further dissociated in the presence of hyaluronidase (0.01%). The resulting cell suspension (95% absorbing enterocytes; viability greater than 90%) was incubated with 14C-labeled substrates to measure 14CO2 production in parallel with substrate disappearance. The capacity to utilize glutamine was high and remained steady during the suckling period. Glucose utilization capacity was limited at birth and increased more than 3-fold during the first week of suckling. Such an increase was not observed in piglets kept unsuckled since birth. Galactose utilization capacity remained steady during the first week but afterward gradually disappeared. Lactate and pyruvate production through glycolysis was the major pathway accounting for glucose or galactose disappearance. A capacity for a net glucose production from galactose was evidenced during the first week of suckling. Thus, isolated newborn pig enterocytes exhibit specific and transient metabolic characteristics during the first postnatal week.

The capacities of viable colonic epithelial cells to metabolize glucose, glutamine and n-butyrate were studied in 30-kg pigs adapted to a high fiber (12% sugar beet fiber) or a low fiber diet. Glucose and glutamine were extensively utilized but predominantly not oxidized, whereas n-butyrate oxidation accounted for 45% of n-butyrate metabolism and was not greatly affected by the presence of glucose or glutamine. With both diets, glycolysis was the major pathway accounting for glucose disappearance. There was a sparing effect of n-butyrate on both glycolysis and glucose oxidation. Moreover, the glycolytic capacity was 25% lower in pigs fed the high fiber diet. Data suggest that 6-phosphofructo-1-kinase could be the regulatory step in glycolysis. Nevertheless, its maximum activity was not affected by the diet or by the presence of n-butyrate. Glutamine metabolism was slightly affected by fiber in the diet and by the presence of n-butyrate. In addition to CO2, butyrate was converted into ketone bodies. Glucose and glutamine did not substantially alter n-butyrate metabolism. We conclude that some metabolic features of pig colonocytes, such as the capacity to oxidize n-butyrate, resemble those of rat and human colonocytes. Moreover, some characteristics, such as the glycolytic capacity, can be modulated by the level of fiber in the diet.

Intestinal mucosal morphometry and ileal epithelial renewal were studied in conventional (CV) and germ-free (GF) rats fed either poorly digestible amylomaize or normal maize starch diets. Intestinal morphometry and position of labelled enterocytes were studied at various times after tritiated thymidine injection. With amylomaize starch diet, no difference was observed in the size of crypts (C), villi (V) and C + V between duodenum and jejunum both in CV and GF rats. In the ileum, however, values were significantly lower than those in the duodenum and jejunum. Furthermore, the presence of the microbial flora led to higher values when compared with GF values. Despite the morphological modifications in the ileum, no significant difference was detected in the labelled cell positions and epithelial renewal time between CV and GF values. This suggests that the resistant part of amylomaize starch was responsible for the modification in mucosal morphometry and the longer ileal epithelium renewal time in CV rats which then becomes similar to that in GF rats.

HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. Energy metabolism in pig colonocytes after adaptation to a high fibre diet B Darcy-Vrillon, Mt Morel, C Cherbuy, F Bernard, Jc Meslin, L Posho, F Blachier, Ph Duée

The influence of low dietary linoleic acid level (an essential fatty acid deficiency) on the intestine mucosal morphology and the purified brush border membrane (BBM) lipid composition was investigated in the rat. Electron micrographs and morphometric measurements showed that villi and crypt sizes as well as the ultrastructure of epithelial cells were altered. Cholesterol (CHOL) and phospholipid (PL) levels, CHOL/PL ratio and PL class distribution were not changed by the low linoleate diet. However, the fatty acid composition of phospholipids was markedly modified in the enterocyte BBM, showing elevated amounts of palmitoleic (16:1n-7), oleic (18:1n-9) and 5,8,11-eicosatrienoic (20:3n-9) acids and, by contrast, depressed linoleic (18:2n-6) and arachidonic (20:4n-6) acid levels. Although the underlying mechanisms remain unknown the results obtained suggest that essential fatty acids (EFA) could be directly involved in the trigger action of the observed alterations, as regards both their dynamic (metabolic) and structural roles.

The aim of this study was to assess the possible modifications in the conventional intestine when deprived of its symbiotic microflora. The experiment was designed to study the effect of a heavy antibiotic dose on fecal microflora during the 33-d treatment period as well as its effects upon the intestinal wall. Conventional adult mice received either a casein-starch diet (conventional controls) or an antibiotic-supplemented (0.66 % dry matter, DM) diet (treated conventionals); Furthermore, germ-free (axenic) mice taken from isolators to the open animal room received the same antibiotic-supplemented diet (treated axenics) Fecal microbial population remained around 10(8)/g in the conventional mice while it decreased to 10(3)/g in the treated conventional mice. Fecal microbial population of the treatedaxenic mice dropped to 10(2)/g. At the end of the 33-d treatment period, no significant difference in ileal villus height between the treated or control groups no difference either was seen in the aspects of the villus and cell surface as shown by scanning electron microscopy. In the control group, however, development of bacterial colonies exhibiting various shapes were observed on the intestinal mucus. Although it was found that antibiotic treatment was followed by significant changes in microbial population and biochemical composition of digestive contents, this study concluded that the structure of the distal ileal epithelium was not impaired.

The aim of this study was to assess the possible modifications in the conventional intestine when deprived of its symbiotic microflora. The experiment was designed to study the effect of a heavy antibiotic dose on fecal microflora during the 33-d treatment period as well as its effects upon the intestinal wall. Conventional adult mice received either a casein-starch diet (conventional controls) or an antibiotic-supplemented (0.66% dry matter, DM) diet (treated conventionals); Furthermore, germ-free (axenic) mice taken from isolators to the open animal room received the same antibiotic-supplemented diet (treated axenics) Fecal microbial population remained around 10(8)/g in the conventional mice while it decreased to 10(3)/g in the treated conventional mice. Fecal microbial population of the treated axenic mice dropped to 10(2)/g. At the end of the 33-d treatment period, no significant difference in ileal villus height between the treated or control groups no difference either was seen in the aspects of the villus and cell surface as shown by scanning electron microscopy. In the control group, however, development of bacterial colonies exhibiting various shapes were observed on the intestinal mucus. Although it was found that antibiotic treatment was followed by significant changes in microbial population and biochemical composition of digestive contents, this study concluded that the structure of the distal ileal epithelium was not impaired.

No information concerning the effect of weaning on intestinal cell proliferation is currently available in large species with early intestinal morphogenesis, a group including most domestic animals and humans. Changes in intestinal morphology and epithelial cell renewal were investigated in 1-, 5-, and 8-wk-old suckling and 8-wk-old weaned lambs after injection of [3H]thymidine. In suckling lambs a gradual increase in crypt depth occurred with age, especially in the proximal intestine, whereas villus height was significantly reduced in the distal regions. At 8 wk of age weaned and prolonged-suckling lambs exhibited no significant differences in crypt depth throughout the intestine and in villus height proximally. However, weaned lambs had shorter villi in the jejunum and ileum. The highest enterocyte migration rates (4.4-9.7 microns/h) were observed in 1-wk-old lambs. In suckling animals, migration rates decreased with age by 60, 51, and 11% in the duodenum, jejunum, and ileum, respectively. Weaned and prolonged-suckling 8-wk-old lambs had a similar rate of enterocyte migration in the ileum. Furthermore, ruminating animals exhibited only slightly higher migration rates in the duodenum and the jejunum (53 and 15%, respectively). In suckling lambs, epithelial cell renewal required 2.1-4.0, 4.5-6.3, and 4.0-5.3 days at 1, 5, and 8 wk of age, respectively, whereas labeled cells reached the tips of the villi within 3.0-3.1 days in weaned animals. These data suggest that the suckling period corresponds to a gradual and important phase of postnatal intestinal adaptation in the sheep, a species with early patterns of intestinal cell replacement.(ABSTRACT TRUNCATED AT 250 WORDS)

The influence of the mode of sterilization of the diet (gamma-irradiation vs. autoclaving) on the histamine and mast cells distribution in the intestinal mucosa, was studied in germ free (GF) and conventional (CV) rats. Interactions between the diet and the digestive microflora were observed. Histamine concentration and mast cells counts are higher in CV rats small intestine than in GF's. The differences are increased with the irradiated diet.

The most striking effect of Clostridium difficile infection is its degrading of the intestinal barrier. The aim of this study is to establish whether the cellular or paracellular constituent of the barrier is the initial target of the toxins produced by C difficile. Accordingly, the caecal epithelium of C3H/He mice was challenged under three experimental conditions with the C difficile strain VPI 10463: (1) by in vivo inoculation of axenic mice, (2) by adding the toxins to ligated caeca in vivo, and (3) by adding them to the mucosal side of isolated caeca in Ussing chambers. Under all three conditions, the epithelial barrier was tested in caeca mounted in these chambers. The transepithelial potential difference (PD), electrical conductance (G), and intact and degraded Horseradish peroxidase (HRP) fluxes were used as indexes of permeability. Results were as follows: (1) In axenic mice, C difficile caused severe infection, produced toxins A and B, reduced PD, and enhanced G and intact HRP fluxes without changing degraded HRP fluxes, (2) four hours after the toxins were added to ligated caeca in vivo, PD was relatively unaltered, but G, and intact and degraded HRP fluxes increased, and (3) when toxins were added to caeca during two hours in the Ussing chambers, the only modification observed was an increase in degraded-HRP fluxes. These results indicate that the C difficile toxins gradually cause intestinal lesions. After an apparent resistance, they stimulate the endocytotic process and then increase paracellular permeability and finally cause loss of cell viability.

This study compares, in 2-d-old rats, the migration rates of epithelial cells on villi of the small intestine, using two labelling methods: a single [3H] thymidine injection; and cytoplasmic labelling by a single ingestion of Pu-citrate. Histoautoradiography showed negligible diffusion of Pu after the initial retention, which was mostly confined to the epithelial cells of the villi. However, after sloughing of labelled cells in the intestinal lumen, Pu was reabsorbed by the distal epithelial cells. In segments in which Pu reabsorption was negligible, the migration rates of Pu- and 3H-labelled cells were very close. These rates, expressed in micrometers, were almost constant along the length of the villus, and the Pu and 3H labelling edges reached the top of the villi in about 5 and 7 d, respectively. Once Pu retention had reached its maximum in 9 equal segments cut along the small intestine, tissue counting showed an exponential Pu release of 30-40%/d from each segment until the end of the experiment at d16. This constant release might reflect a constant cell migration rate during the period from Pu ingestion until d16.

The most striking effect of Clostridium difficile infection is its degrading of the intestinal barrier. The aim of this study was to characterize the effect of C. difficile toxins on the epithelial layer including cellular and paracellular constituents. Accordingly, the caecum of C3H/He mice was challenged under 3 experimental conditions with C. difficile strain VP1 10463 : 1) by in vivo inoculation of axenic mice, 2) by adding the toxins to ligated caeca in vivo, and 3) by adding them to the mucosal side of isolated caeca in Ussing chambers. Under all 3 conditionals, the epithelial barrier was tested in caeca mounted in these chambers. The transeptithelial potential difference (PD), electrical conductance (C), and intact and degraded Horseradish peroxidase (HRP) fluxes were used as indexes of permeability. Results : 1) In axenic mice, C. difficile caused severe infection, produced toxins A and B, reduced PD, and enhanced C and intact HRP fluxes without changing degraded HRP fluxes; 2) 4 hours after the toxins were added to ligated caeca in vivo, PD was relatively unaltered, but C intact and degraded HRP fluxes increased, and 3) when toxins were added to caeca during 2 hours in the Ussing chambers, the only modification observed was an increase in degraded-HRP fluxes. These results indicate that the C. difficile toxins gradually cause intestinal lesions. After an apparent resistance, they stimulate the endocytotic process, increase paracellular permeability and finally cause loss of cell viability.