[Effect of dietary magnesium levels on cardiac lesions in rats fed a diet rich in rapeseed oil]. / Effet à moyen terme de la teneur en magnésium d'un régime riche en huile de colza sur l'apparition des lésions du myocarde chez le rat

Cardiac lesions (histiocyte infiltration and fibrosis) obtained in rats fed rapeseed oil and 3 magnesium levels (high, low and normal) during 30 or 50 days are quantified by their size. Correspondence factorial analysis is used to compare the results. After 30 days, less cardiac lesions of histiocyte infiltration or fibrosis appear with rats fed a diet high in both rapeseed oil and magnesium than with normal or low magnesium diet. After 50 days, cardiopathogenic effects are less severe with normal magnesium diet than with the others (high or low). In both cases, more severe lesions are seen with low magnesium diet. It is concluded that magnesium overload in the diet does not protect against the cardiopathogenic effects of a diet containing high levels of rapeseed oil. Classical nephrocalcinosis and hypomagnesemia are seen with a low magnesium diet (70 ppm).

Regional cholesterol synthesis in the intestinal mucosa of the genetically hypercholesterolaemic RICO rat: kinetic study following whole-body gamma-irradiation.

PURPOSE: To investigate regional cholesterol synthesis and kinetics following whole-body gamma-irradiation in the genetically hypercholesterolaemic RICO rat. MATERIALS AND METHODS: Male RICO rats were fed a semi-purified diet for 1 month. At 10 weeks old they were exposed to gamma-irradiation (4 Gy, 1.5 Gy/min) together with controls. At intervals from 1-8 days after irradiation an intraperitoneal administration of [1-14C] acetate was given in order to estimate cholesterogenesis in mucosal cells located at different sites in the small intestine. The protein and DNA contents of the different enterocytes isolated along the crypt/villus axis in four equal parts of the intestine were also determined. RESULTS: A marked decrease of the mean quantities of cholesterol, DNA or protein in mucosa was seen 1 and 2 days after irradiation, showing the loss of 30-40% of the intestinal epithelium. An overshoot of the cell amount was observed after 4 days with a return to basal values by 8 days after irradiation. The kinetic and topological evolution of cholesterol radioactivity, which reflects in situ cholesterol synthesis, showed a typical gradient in controls and at 8 days after irradiation. Cholesterogenesis decreased from the first to the third quarter of the small intestine (duodenum to proximal ileum), and then increased in the fourth quarter (distal ileum). In all segments of the small intestine, cholesterogenesis decreased from crypt cells to villus tip. At days 1 and 2 the gradient of cholesterogenesis on the villus was abolished. A slow recovery was seen from day 4 with a strong overshoot of cholesterol synthesis in crypt cells in every part of the small intestine. CONCLUSIONS: The RICO rat is a useful model for studying the effect of irradiation on regional cholesterogenesis in intestinal mucosa. Cholesterol synthesis in crypt cells was lowered 1 and 2 days after irradiation, over-expressed after 4 days and subsequently returned to its normal level.

Effects of microflora on the dimensions of enterocyte microvilli in the rat.

The length and diameter of enterocyte microvilli at mid-villus position were measured on electron-micrographs. The duodenum, jejunum and ileum of axenic (germfree) and holoxenic (conventional) inbred rats fed the same diet have been studied. The microvilli were significantly shorter in all these intestinal regions when the microflora was present. The decrease in microvillus length (due to the presence of microflora), expressed as a percentage of the length in axenic rat, was 5% in the duodenum, 9% in the jejunum and 18% in the ileum. This was not true of microvillus diameter where only the values found for the ileum of axenic rat were significantly lower, the others not differing among themselves. In conclusion, the presence of a total microflora led to a decrease in the length of enterocyte microvilli. These results have been discussed in relation with studies on renewal and enzyme activity in the intestinal epithelium.

Changes in small intestinal mucosa morphology and cell renewal in suckling, prolonged-suckling, and weaned lambs.

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)

[Selective determination of different types of gastrointestinal mucins:use of histochemical reagents]. / Mise au point d'un dosage sélectif des différents types de mucines gastro-intestinales: utilisation de réactifs histochimiques.

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.

[Decrease of the intestinal microflora without consequences for the morphometry and topography of the distal ileum in the mouse treated with antibiotics]. / Diminution de la microflore intestinale sans conséquences sur la morphométrie et la topographie de l'iléon distal chez la souris traitée aux antibiotiques.

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.

Selective in vitro degradation of the sialylated fraction of germ-free rat mucins by the caecal flora of the rat.

Mucins, which are synthesized throughout the gastrointestinal tract, may be degraded by the microflora of the large intestine. The present study was undertaken to determine the differential fate of the various types of mucins. Mucins from germ-free rats were incubated in vitro in the presence of whole caecal flora from the conventional rat. Neutral, acidic and acidic sulphated mucins were spectrophotometrically assayed over time upon anaerobic incubation. Sialylated mucins were more rapidly degraded (90%) than the other two types after 1 h and almost completely within 4 h. Neutral and acidic sulphated mucins, with a 10-fold and 30-fold lower content than the sialylated fraction in the original substrate, were more slowly degraded and to a lesser extent within 4 h, (55 and 40%, respectively). The method used in the present study made it possible to investigate the activity of gut bacteria towards the various types of mucins. The degradation of the three mucin types was not uniform, the highest rate and extent of degradation being observed for sialylated mucins.

Differential influence of butyrate concentration on proximal and distal colonic mucosa in rats born germ-free and associated with a strain of Clostridium paraputrificum.

In vivo influence of butyrate in colonic mucosa was studied using a model of gnotobiotic rats monoassociated with a Clostridium paraputrificum. Rats were fed a diet containing increasing amounts of non-digestible carbohydrates, the fermentation of which led to modulated amounts of butyrate in the large intestine. In the proximal colon, the increase in the butyrate concentration alters crypt depth and the number of mucus-containing cells; the increase in butyrate was highly correlated with the number of neutral-mucin-containing cells. Conversely, in the distal colon, no relation was found between the increase in butyrate concentration and crypt depth or number of mucin-containing cells. In both the proximal and distal colon, the mitotic index remained unchanged. In conclusion, in vivo production of physiological quantities of butyrate had a trophic effect on proximal colonic mucosa, but did not influence the distal epithelium.

Histamine and mast cell distribution in the intestinal wall of the germ free and conventional rats. Influence of the mode of sterilization of the diet.

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. At the opposite in the hindgut, these values are higher in GF than in CV rats, especially in the rats fed the steam sterilized diet. The variations in the wall histamine contents and in the mucosal mast cells counts due to the diet and/or the microflora do not appear to be always correlated.

Intestinal mucosal morphometry and ileal epithelial renewal in conventional and germ-free rats fed an amylomaize starch 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.

Intestinal mucin distribution in the germ-free rat and in the heteroxenic rat harbouring a human bacterial flora: effect of inulin in the diet.

A colorimetric method was used on water-soluble mucin 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 human flora (GF rats associated 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 mucin extract. Each mucin 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 mucin was present and neutral and sulphomucin proportions were of the same order of magnitude. Inulin increased the amount of neutral mucin in the caecal contents and of sulphated mucins in the colonic contents and increased the amounts of neutral and acidic mucins in the caecal mucosa. Mucin distribution in the HE rats was very different from that in the GF rats: the caecal contents contained a high proportion of acidic mucins and very little sulphomucin. The same distribution of mucins was observed in the colonic contents. The caecal mucosa contained less acidic mucin and more sulphomucin than the caecal contents. Inulin decreased acidic mucins and increased sulphated mucins in the caecal contents and increased neutral and sulphated mucins in the colonic contents. Inulin increased sulphomucin in the caecal mucosa and decreased acidic mucin 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, mucin was extensively released from the mucosa to the colonic lumen. This might be related to the bacterial metabolites produced.

Effect of various modifications in the diet on ileal epithelium renewal in germ-free and conventional rats.

Renewal of the ileal epithelium has been studied in germ-free (GF) and conventional (CV) rats fed with a semi-synthetic diet in the following forms: a) pellets sterilized by autoclaving (Pel. Au. diet); b) powder sterilized by gamma irradiation; when given to the rats, its weight in water was added to make a paste (Pa. I. diet); c) powder treated as (b) but with 10 p. 100 lactose added (L. Pa. I. diet). Ileal epithelium renewal time was similar in the GF rats fed with the Pel. Au. and Pa. I. diets. It was shorter in the CV rats fed with these two diets than in the GF rats and also in the CV rats given the Pa. I. diet than in those given the Pel. Au. diet. In GF and CV rats fed with the lactose diet, L. Pa. I., ileal epithelium renewal time was similar. These different effects were mainly due to variations in the number of cells produced in Lieberkühn's crypts. They were not related to changes in the intestinal bile salts pool caused by the different diet treatments.

Variation of mucin distribution in the rat intestine, caecum and colon: effect of the bacterial flora.

The influence of the intestinal microflora on mucin types was studied in the small intestine, caecum and colon of conventional (CV) rats as compared to germ-free (GF) rats. A colorimetric method was used on purified water-soluble mucin extracted from mucosal scrapings and contents. Variations occurred between the three anatomical sites both in the mucosas and intestinal contents of GF rats. In CV rats, the presence of the bacterial flora led to different effects depending on the intestinal site: in the small intestinal mucosa, neutral and sulphomucins values were higher whereas sialomucin was much lower. Conversely, sialomucin was higher in the caecal and colonic mucosas and contents whereas sulphated mucins were decreased significantly in caecal contents and caecal and colonic mucosas. These variations in the contents may reflect the bacterial mucolytic activity and the effect of bacterial metabolites on the mucosa.

Epithelial cell migration on small intestinal villi in the neonatal rat. Comparison between [3H] thymidine and cytoplasmic labelling after Pu-citrate ingestion.

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.

Intestinal absorption of macromolecules during viral enteritis: an experimental study on rotavirus-infected conventional and germ-free mice.

Epithelial transport and degradation of horseradish peroxidase (HRP), a macromolecular tracer, was studied in conventional and germ-free suckling mice following an experimental infection with rotavirus. Conventional and germ-free mice developed diarrhea from days 2 to 8 postinfection (pi), with growth failure. In mucosal homogenates, infectious virus detected by immunofluorescence on MA 104 cells was present from day 2 through day 8 pi in germ-free mice, but persisted longer (day 13 pi) in conventional mice. Only mild histological lesions were observed during diarrhea, but obvious macrovacuolation of epithelial cells and increased cellular density occurred during the convalescence period (days 9 to 13 pi). Intact and degraded HRP fluxes from mucosa to serosa were measured in vitro on segments of jejunum mounted in Ussing chambers. Both groups of mice developed increased HRP permeability during the experimental period, but at different times after inoculation: during the diarrheal period (days 2 and 3 pi) conventional mouse epithelium absorbed five times more HRP than noninfected controls and during the convalescence period (days 9 to 13 pi) HRP absorption in germ-free mice rose 10-fold as compared to its level before infection. In both cases, this increase in HRP permeability was entirely due to an increase in intact HRP absorption, probably via a transcellular route, and occurred without any alteration in degraded HRP transport. These results indicate that in mice, rotavirus infection causes a transient rise in gut permeability to undegraded proteins. The intestinal microflora seems to affect the timing, magnitude, and duration of this increased permeability.

Histamine and mast cell distribution in the gastrointestinal wall of the rat: comparison between germ-free and conventional rats.

The distributions of histamine and mast cells in the intestinal mucosa have been studied in conventional (CV) and germ-free (GF) rats. Both distributions do not appear to be uniform throughout the gastrointestinal tract. The presence of digestive microflora influences these distributions: more histamine and mucosal mast cells are present in the small intestine of CV than in GF rats. Paradoxically the opposite is observed in the large intestine. Differences in mast cell counts in the crypt area of the small intestine (higher in CV than in GF rats) confirm the role of microflora on crypt cell proliferation due to a mild chronic inflammation. In the large intestine, however, the microflora appears to have a more complex influence and could act indirectly on histamine turnover.

Effects of a low dietary linoleic acid level on intestinal morphology and enterocyte brush border membrane lipid composition.

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.

Colonic mucin discharge by a cholinergic agonist, prostaglandins, and peptide YY in the isolated vascularly perfused rat colon.

UNLABELLED: 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 x 10(-6) to 2 x 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 x 10(-4) M versus 28.8 +/- 1.5 for controls (n = 6). After infusion of 1.25 and 2.5 microM 16,16-dimethyl prostaglandin E2 (dmPGE2), 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 microM dmPGE2, respectively, vs. 12.3 +/- 4.1% for controls). In contrast, prostaglandin F2alpha 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 dmPGE2. 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.

Evolution of the caecal epithelial barrier during Clostridium difficile infection in the mouse.

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.